Gene therapy for mucopolysaccharidosis iiib

ABSTRACT

Provided herein is a recombinant AAV (rAAV) comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises an AAV 5′ inverted terminal repeat (ITR), an engineered nucleic acid sequence encoding a functional human N-acetyl-alpha-glucosaminidase (hNAGLU), a regulatory sequence which direct expression of hNAGLU in a target cell, and an AAV 3′ ITR. Also provided is a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer, and a method of treating a human subject diagnosed with MPS IIIB.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/768,547, filed May 29, 2020, which is a national stage applicationunder 35 U.S.C. 371 of PCT/US2018/063166, filed Nov. 29, 2018, nowexpired, which claims the benefit of U.S. Patent Application No.62/593,090, filed Nov. 30, 2017, now expired. These applications areincorporated by reference in their entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Applicant hereby incorporates by reference the Sequence Listing materialfiled in electronic form herewith. This file is labeled“UPN-18-8482.US.C1_ST26_Sequence Listing.xml” (Created Jun. 28, 2023,26,200 bytes).

BACKGROUND OF THE INVENTION

Mucopolysaccharidosis type IIIB (MPS IIIB, or Sanfilippo syndrome typeB, Sanfilippo type B disease), is an autosomal recessive inheriteddisorder caused by the deficiency of the enzymeN-acetyl-alpha-D-glucosaminidase (NAGLU) involved in the lysosomalcatabolism of the glycosaminoglycans (GAG) heparan sulfate. Thisdeficiency leads to the intracellular accumulation of undegraded heparansulfate as well as gangliosides GM2 and GM3 in the central nervoussystem causing neuronal dysfunction and neuroinflammation.

MPS IIIB is a neurodegenerative disorder characterized by an initialsymptom free period followed by progressive intellectual decline,finally resulting in severe dementia. Severe behavioral problems are apredominant symptom in most patients, characterized mainly by extremelyhyperactive behavior. Other symptoms include sleeping problems,recurrent diarrhea, frequent ear, nose and throat infections, hearingand visual impairment and epilepsy. Patients usually die at the end ofthe second or the beginning of the third decade of life, although longersurvival has been reported in patients with an attenuated form of MPSIIIB.

There is no specific treatment for MPS IIIB. Clinical management ofpatients with MPS IIIB currently still consists mainly of supportivecare, aimed at ameliorating symptoms and prevention of complications.Medications are used to relieve symptoms (such as anticonvulsants forseizures) and improve quality of life. Hematopoietic stem celltransplantation, such as bone marrow transplantation or umbilical cordblood transplantation, does not seem to ameliorate neuropsychologicaldeterioration significantly. Enzyme replacement therapies (ERT) for MPSIIIB via intravenous administration and intracerebroventricular infusionshows elevated enzyme activity of NAGLU in murine models and arecurrently under investigation in clinical trials on MPS IIIB patients.Still, ERT requires multiple administrations, significantly impactspatient quality of life, and is at a high expense. See, e.g.,Aoyagi-Scharber M et al, Clearance of Heparan Sulfate and Attenuation ofCNS Pathology by Intracerebroventricular BMN 250 in Sanfilippo Type BMice, Mol Ther Methods Clin Dev. 2017 Jun. 6; 6:43-53. doi:10.1016/j.omtm.2017.05.009.eCollection 2017 Sep. 15; and WO2017132675A1.

A need in the art exists for compositions and methods for efficienttreatment of MPS IIIB.

SUMMARY OF THE INVENTION

In one aspect, provided herein is a vector comprising an engineerednucleic acid sequence encoding a functional humanN-acetyl-alpha-glucosaminidase (hNAGLU) and a regulatory sequence whichdirect expression thereof in a target cell. In one embodiment, thehNAGLU coding sequence is at least 95% identical to SEQ ID NO: 1. In afurther embodiment, the hNAGLU coding sequence is SEQ ID NO: 1.

In another aspect, provided is a recombinant AAV (rAAV) comprising anAAV capsid and a vector genome packaged therein, wherein the vectorgenome comprises an AAV 5′ inverted terminal repeat (ITR), an engineerednucleic acid sequence encoding a functional hNAGLU, a regulatorysequence which direct expression of hNAGLU in a target cell, and an AAV3′ ITR. In one embodiment, the hNAGLU coding sequence is at least 95%identical to SEQ ID NO: 1. In a further embodiment, the hNAGLU codingsequence is SEQ ID NO: 1. In yet a further embodiment, the AAV vectorgenome comprises the sequence of SEQ ID NO: 4 (AAV.CB7.CI.hNAGLUco.rBG).In some embodiments, the AAV capsid is an AAV9 capsid. In oneembodiment, the rAAV (AAV9.CB7.CI.hNAGLUco.rBG) comprises an AAV9 capsidand a vector genome comprising the sequence of SEQ ID NO: 4.

In yet another aspect, a pharmaceutical composition comprising a rAAV ina formulation buffer is provided, wherein the rAAV comprises an AAVcapsid and a vector genome packaged therein, wherein the vector genomecomprises an AAV 5′ inverted terminal repeat (ITR), an engineerednucleic acid sequence encoding a functional hNAGLU, a regulatorysequence which direct expression of hNAGLU in a target cell, and an AAV3′ ITR.

In a further aspect, a method of treating a human subject diagnosed withMPS IIIB is provided. The method comprises administering to a subject inneed a suspension of a rAAV as described herein in a formulation buffer.

Further provided are an engineered nucleic acid sequence comprising anengineered sequence of SEQ ID NO: 1 or a sequence 95% identical theretoand an expression cassette comprising an engineered nucleic acidsequence encoding a functional hNAGLU, and a regulatory sequence whichdirect expression thereof. In one embodiment, the hNAGLU coding sequenceis at least 95% identical to SEQ ID NO: 1.

Other aspects and advantages of the invention will be readily apparentfrom the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that there is NAGLU activity in brain, spinal cord,liver, heart, and serum 3 months after intracerebroventricular (icv)administration of AAV9.CB7.CI.NAGLUco.rBG. There is a dose dependentincrease in NAGLU activity in the brain, spinal cord, liver, and heart.There is essentially no activity in the low dose animals, partial rescuein the mid dose, and activity levels equal or above the heterozygous inall organs from the high dose treated animals. Activity in the serum isvery low due to the presence of anti hNAGLU circulating antibodies.

FIGS. 2A and 2B provide lysosomal storage assessed by LIMP2immunostaining (FIG. 2A) and quantification (FIG. 2B) in brain 3 monthsafter intracerebroventricular administration ofAAV9.CB7.CI.hNAGLUco.rBG. LIMP2 immunostaining of lysosomal membranesshow a reduction of the storage burden at the high dose (one way AnovaKruskall Wallis test with post hoc Dunn's multiple comparison test,alpha 0.05).

FIGS. 3A and 3B provide immunohistochemical staining (FIG. 3A) andhistopathology cumulative score (FIG. 3B) in brain 3 months after ICVadministration of AAV9.CB7.CI.hNAGLUco.rBG. Brain score is thecumulative sum of 4-grade severity scores of glial cell vacuolation inbrain, neuronal vacuolation in brain cortex, neuronal vacuolation inbrainstem and hindbrain, perivascular mononuclear cell infiltrationmononuclear cell infiltration (maximum score of 20). Low dose MPS IIIbmice are similar to vehicle-treated whereas both mid dose and high dosetreated mice have a decreased neuropathology score in the brain andspinal cord (spinal cord not shown). The correction of neuropathology isstatistically significant in the mid- and high dose groups treatedanimals (one way Anova Kruskall Wallis test with post hoc Dunn'smultiple comparison test, alpha 0.05).

FIG. 4 shows neurologic function assessed by the rocking rotarod 2months after ICV administration of AAV9.CB7.CI.hNAGLU.rBG. Mice arepositioned on a rotating rod (10 revolutions per minutes) with aninversion of the rotation direction after each revolution. The latencyto fall is measured over a maximum period of 180 seconds during 3consecutive assays. The mean latency of the 3 assays is reported as anindicator of balance and coordination. Vehicle-treated MPS IIIb micepresent a neurologic deficit that cause them to fall from the rotatingrod before heterozygous mice. High-dose treated mice tend to performbetter than the untreated but statistical significance is not reacheddue to interindividual variability.

FIG. 5 provides a grading scale used to assess the clinical health ofmice used in studies to determine long-term effects ofAAV9.CB7.CI.hNAHLU.rBG treatment.

DETAILED DESCRIPTION OF THE INVENTION

Compositions useful for the treatment of Mucopolysaccharidosis type IIIb(MPS IIIB) and/or alleviating symptoms of MPSIIIB are provided herein.These compositions comprise a nucleic acid sequence encoding afunctional human N-acetyl-alpha-D-glucosaminidase (hNAGLU) and aregulatory sequence which direct expression thereof in a target cell,wherein the hNAGLU coding sequence is at least 95% identical to SEQ IDNO: 1.

In one embodiment, the compositions and methods described herein involvenucleic acid sequences, expression cassettes, vectors, recombinantviruses, other compositions and methods for expression of a functionalhuman NAGLU (hNAGLU). In another embodiment, the compositions andmethods described herein involve nucleic acid sequences, expressioncassettes, vectors, recombinant viruses, host cells, other compositionsand methods for production of a composition comprising the nucleic acidsequence encoding a functional hNAGLU. In yet another embodiment, thecompositions and methods described herein involve nucleic acidsequences, expression cassettes, vectors, recombinant viruses, othercompositions and methods for delivery of the nucleic acid sequenceencoding a functional hNAGLU to a subject for the treatment of MPS IIIB.In one embodiment, the compositions and methods described herein areuseful for providing a therapeutic level of NAGLU into the centralnervous system (CNS). Additionally or alternatively, the compositionsand methods described herein are useful for providing a therapeuticlevel of NAGLU in the periphery, such as, e.g., blood, liver, kidney, orperipheral nervous system. In certain embodiments, an adeno-associatedviral (AAV) vector-based method described herein provides a newtreatment option, helping to restore a desired function of NAGLU, toalleviate a symptom associated with MPS IIIB, to improve MPSIIIB-related biomarkers, or to facilitate other treatment(s) for MPSIIIB, by providing expression of NAGLU protein in a subject in need.

As used herein, the term “a therapeutic level” means an enzyme activityat least about 5%, about 8%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, about 100%, more than 100%, about 2-fold, about 3-fold, orabout 5-fold of a healthy control. Suitable assays for measuring NAGLUenzymatic activity are described herein. In some embodiments, suchtherapeutic levels of NAGLU may result in alleviation of the MPS IIIBrelated symptom(s); improvement of MPS IIIB-related biomarkers ofdisease; or facilitation of other treatment(s) for MPS IIIB, e.g., GAGlevels in the cerebrospinal fluid (CSF), serum, urine or any otherbiological samples; prevention of neurocognitive decline; reversal ofcertain MPS IIIB-related symptoms and/or prevention of progression ofMPS IIIB-related certain symptoms; or any combination thereof.

As used herein, “a healthy control” refers to a subject or a biologicalsample therefrom, wherein the subject does not have an MPS disorder. Thehealthy control can be from one subject. In another embodiment, thehealthy control is a pool of multiple subjects.

As used herein, the term “biological sample” refers to any cell,biological fluid or tissue. Suitable samples for use in this inventionmay include, without limitation, whole blood, leukocytes, fibroblasts,serum, urine, plasma, saliva, bone marrow, cerebrospinal fluid, amnioticfluid, and skin cells. Such samples may further be diluted with saline,buffer or a physiologically acceptable diluent. Alternatively, suchsamples are concentrated by conventional means.

With regard to the description of these inventions, it is intended thateach of the compositions herein described, is useful, in anotherembodiment, in the methods of the invention. In addition, it is alsointended that each of the compositions herein described as useful in themethods, is, in another embodiment, itself an embodiment of theinvention.

Unless defined otherwise in this specification, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs and byreference to published texts, which provide one skilled in the art witha general guide to many of the terms used in the present application.

As used herein, “disease”, “disorder” and “condition” areMucopolysaccharidosis type IIIb (MPS IIIB, MPS IIIb, also known asSanfilippo syndrome type B or Sanfilippo type B disease).

As used herein, the term “MPS IIIB-related symptom(s)” or “symptom(s)”refers to symptom(s) found in MPS IIIB patients as well as in MPS IIIBanimal models. Such symptoms include but not limited to delayed speech;difficulty with social interactions and communication; sleepdisturbances; progressive intellectual disability and the loss ofpreviously acquired skills (developmental regression); seizures andmovement disorders; a large head; a slightly enlarged liver (mildhepatomegaly); a soft out-pouching around the belly-button (umbilicalhernia) or lower abdomen (inguinal hernia); short stature, jointstiffness, mild dysostosis multiplex, multiple skeletal abnormalities;chronic diarrhea; recurrent upper respiratory infections; recurrent earinfections; hearing impairment; vision problems; Asymmetric septalhypertrophy; Coarse facial features; Coarse hair; Dense calvaria;Dysostosis multiplex; Growth abnormality; Heparan sulfate excretion inurine; GAG accumulation in the cerebrospinal fluid (CSF), serum, urineand/or other biological samples; abnormal expression and/or enzymeactivity of N-sulfoglycosamine sulfohydrolase (SGSH) orN-sulfoglycosamine sulfohydrolase (IDUA); accumulation of GM2 and GM3;changed activity in lysosomal enzymes; accumulation of free unesterifiedcholesterol in the CNS; inflammatory response in the CNS and skeletaltissues; excess hair growth (Hirsutism); Hyperactivity; Ovoidthoracolumbar vertebrae; Splenomegaly; Synophrys; Thickened ribs;hernias; and a wobbly and erratic walk.

“Patient” or “subject” as used herein means a male or female human,dogs, and animal models used for clinical research. In one embodiment,the subject of these methods and compositions is a human diagnosed withMPS IIIB. In certain embodiments, the human subject of these methods andcompositions is a prenatal, a newborn, an infant, a toddler, apreschool, a grade-schooler, a teen, a young adult or an adult. In afurther embodiment, the subject of these methods and compositions is apediatric MPS IIIB patient.

Clinical examination and urine tests (excess mucopolysaccharides areexcreted in the urine) are the first steps in the diagnosis of an MPSdisease. Enzyme assays measuring levels of enzyme activity in the blood,skin cells or a variety of cells are also used to provide definitivediagnosis of MPS IIIB. See,www_ncbi_nlm_nih_gov/gtr/all/tests/?-term=4669[geneid]; andwww_ncbi_nlm_nih_gov/gtr/all/tests/?term=C0086648-[DISCUI]&filter=method:1_2;testtype:clinical. Various genetic testing detecting a mutation of NAGLUassociated with MPS IIIB is available. See, e.g.,www_ncbi_nlm_nih_gov/gtr/conditions/C0086648/;www_ncbi_nlm_nih_gov/gtr/all/-tests/?term=C0086648[DISCUI]&filter=method:2_7;testtype:clinical; and www_ncbi_nlm_nih_gov/gtr/tests/506481/. Prenataldiagnosis using amniocentesis and chorionic villus sampling can verifyif a fetus is affected with the disorder. Genetic counseling can helpparents who have a family history of the mucopolysaccharidoses determineif they are carrying the mutated gene that causes the disorders. See,e.g., A Guide to Understanding MPS III, National MPS Society, 2008,mpssociety_org/learn/diseases/mps-iii/.

“Comprising” is a term meaning inclusive of other components or methodsteps. When “comprising” is used, it is to be understood that relatedembodiments include descriptions using the “consisting of” terminology,which excludes other components or method steps, and “consistingessentially of” terminology, which excludes any components or methodsteps that substantially change the nature of the embodiment orinvention. It should be understood that while various embodiments in thespecification are presented using “comprising” language, under variouscircumstances, a related embodiment is also described using “consistingof” or “consisting essentially of” language.

It is to be noted that the term “a” or “an”, refers to one or more, forexample, “a vector”, is understood to represent one or more vector(s).As such, the terms “a” (or “an”), “one or more,” and “at least one” isused interchangeably herein.

As used herein, the term “about” means a variability of plus or minus10% from the reference given, unless otherwise specified.

1. N-acetyl-alpha-glucosaminidase (NAGLU)

As used herein, the terms “N-acetyl-alpha-glucosaminidase”, “NAGLU” and“NaGlu” are used interchangeably with “Alpha-N-Acetylglucosaminidase”.The invention includes any variant of NAGLU protein expressed from thenucleic acid sequences provided herein, or a functional fragmentthereof, which restores a desired function, ameliorates a symptom,improves symptoms associated with a MPS IIIB-related biomarker, orfacilitates other treatment(s) for MPS IIIB when delivered in acomposition or by a method as provided herein. Examples of a suitablebiomarker for MPSIII include that described in WO 2017/136533, which isincorporated herein by reference.

As used herein, the term “functional NAGLU” means an enzyme having theamino acid sequence of the full-length wild-type (native) human NAGLU(as shown in SEQ ID NO: 2 and UniProtKB accession number: P54802), avariant thereof, a mutant thereof with a conservative amino acidreplacement, a fragment thereof, a full-length or a fragment of anycombination of the variant and the mutant with a conservative amino acidreplacement, which provides at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 90%, or about the same, or greater than 100% of thebiological activity level of normal human NAGLU. In one embodiment, afunctional NAGLU refers to a wild-type NAGLU protein with sequence ofSEQ ID NO: 2.

Examples of NAGLU variants include but not limited to, E705K, whichconsists of the amino acid sequence of SEQ ID NO: 2 with a Lysine (Lys,K) at the 705th amino acid instead of Glutamic acid (Glu, E) in thewild-type.

As used herein, the “conservative amino acid replacement” or“conservative amino acid substitutions” refers to a change, replacementor substitution of an amino acid to a different amino acid with similarbiochemical properties (e.g. charge, hydrophobicity and size), which isknown by practitioners of the art. Also see, e.g. French et al. What isa conservative substitution? Journal of Molecular Evolution, March 1983,Volume 19, Issue 2, pp 171-175 and YAMPOLSKY et al. The Exchangeabilityof Amino Acids in Proteins, Genetics. 2005 August; 170(4): 1459-1472,each of which is incorporated herein by reference in its entirety.

A variety of assays exist for measuring NAGLU expression and activitylevels by conventional methods. See, e.g., Example 1 as describedherein;www_ncbi_nlm_nih_gov/gtr/all/tests/?term=C0086648[DISCUI]&filter=method:1_2;testtype:clinical; www_ncbi_nlm_nih_gov/gtr/all/tests/?term=C0086648[DISCUI]&filter=method:1_1; testtype:clinical; Kan S H et al, Deliveryof an enzyme-IGFII fusion protein to the mouse brain is therapeutic formucopolysaccharidosis type IIIB. Proc Natl Acad Sci USA. 2014 Oct. 14;111(41):14870-5. doi: 10.1073/pnas.1416660111. Epub 2014 Sep. 29; US2017/0088859; each of which is incorporated by reference herein in itsentirety.

In one aspect, a nucleic acid sequence which encodes a functional NAGLUprotein is provided. In one embodiment, the nucleic acid sequence is thewild-type coding sequence reproduced in SEQ ID NO: 3. In one embodiment,the nucleic acid sequence is at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80% identicalthereto the wild-type human NAGLU sequence of SEQ ID NO: 3.

A nucleic acid refers to a polymeric form of nucleotides and includesRNA, mRNA, cDNA, genomic DNA, peptide nucleic acid (PNA) and syntheticforms and mixed polymers of the above. A nucleotide refers to aribonucleotide, deoxynucleotide or a modified form of either type ofnucleotide (e.g., a peptide nucleic acid oligomer). The term alsoincludes single- and double-stranded forms of DNA. The skilled man willappreciate that functional variants of these nucleic acid molecules arealso intended to be a part of the present invention. Functional variantsare nucleic acid sequences that can be directly translated, using thestandard genetic code, to provide an amino acid sequence identical tothat translated from the parental nucleic acid molecules.

In certain embodiments, the nucleic acid molecules encoding a functionalhuman NAGLU (hNAGLU), and other constructs encompassed by the presentinvention and useful in generating expression cassettes and vectorgenomes may be engineered for expression in yeast cells, insect cells ormammalian cells, such as human cells. Methods are known and have beendescribed previously (e.g. WO 96/09378). A sequence is consideredengineered if at least one non-preferred codon as compared to a wildtype sequence is replaced by a codon that is more preferred. Herein, anon-preferred codon is a codon that is used less frequently in anorganism than another codon coding for the same amino acid, and a codonthat is more preferred is a codon that is used more frequently in anorganism than a non-preferred codon. The frequency of codon usage for aspecific organism can be found in codon frequency tables, such as inwww.kazusa.jp/codon. Preferably more than one non-preferred codon,preferably most or all non-preferred codons, are replaced by codons thatare more preferred. Preferably the most frequently used codons in anorganism are used in an engineered sequence. Replacement by preferredcodons generally leads to higher expression. It will also be understoodby a skilled person that numerous different nucleic acid molecules canencode the same polypeptide as a result of the degeneracy of the geneticcode. It is also understood that skilled persons may, using routinetechniques, make nucleotide substitutions that do not affect the aminoacid sequence encoded by the nucleic acid molecules to reflect the codonusage of any particular host organism in which the polypeptides are tobe expressed. Therefore, unless otherwise specified, a “nucleic acidsequence encoding an amino acid sequence” includes all nucleotidesequences that are degenerate versions of each other and that encode thesame amino acid sequence. Nucleic acid sequences can be cloned usingroutine molecular biology techniques, or generated de novo by DNAsynthesis, which can be performed using routine procedures by servicecompanies having business in the field of DNA synthesis and/or molecularcloning (e.g. GeneArt, GenScript, Life Technologies, Eurofins).

In one aspect, the NAGLU coding sequence is an engineered sequence. Inone embodiment, the engineered sequence is useful to improve production,transcription, expression or safety in a subject. In another embodiment,the engineered sequence is useful to increase efficacy of the resultingtherapeutic compositions or treatment. In a further embodiment, theengineered sequence is useful to increase the efficacy of the functionalNAGLU protein being expressed, but may also permit a lower dose of atherapeutic reagent that delivers the functional protein to increasesafety.

In one embodiment, the engineered NAGLU coding sequence is characterizedby improved translation rate as compared to wild-type NAGLU codingsequences. In one embodiment, the NAGLU coding sequence has less than82% identical to the wild-type hNAGLU sequence of SEQ ID NO: 3. In oneembodiment, the NAGLU coding sequence shares less than about 99%, lessthan about 98%, less than about 97%, less than about 96%, less thanabout 95%, less than about 94%, less than about 93%, less than about92%, less than about 91%, less than about 90%, less than about 89%, lessthan about 88%, less than about 87%, less than about 86%, less thanabout 85%, less than about 84%, less than about 83%, less than about82%, less than about 81%, less than about 80%, less than about 79%, lessthan about 78%, less than about 77%, less than about 76%, less thanabout 75%, less than about 74%, less than about 73%, less than about72%, less than about 71%, less than about 70%, less than about 69%, lessthan about 68%, less than about 67%, less than about 66%, less thanabout 65%, less than about 64%, less than about 63%, less than about62%, less than about 61% or less identity to the wild type NAGLU codingsequence. In another embodiment, the NAGLU coding sequence shares about99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%,about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%,about 79%, about 78%, about 77%, about 76%, about 75%, about 74%, about73%, about 72%, about 71%, about 70%, about 69%, about 68%, about 67%,about 66%, about 65%, about 64%, about 63%, about 62%, about 61% or lessidentity to the wild type NAGLU coding sequence. In one embodiment,provided is an engineered nucleic acid sequence comprising a sequence ofSEQ ID NO: 1. In one embodiment, provided herein is an engineerednucleic acid sequence of SEQ ID NO: 1, or a nucleic acid sequence atleast about 95% identical thereto, encoding a functional hNAGLU. Inanother embodiment, the NAGLU coding sequence is at least about 80%, atleast about 81%, at least about 82%, at least about 83%, at least about84%, at least about 85%, at least about 86%, at least about 87%, atleast about 88%, at least about 89%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99% identity to SEQ ID NO: 1, wherein the sequenceencodes a functional hNAGLU.

By “engineered” is meant that the nucleic acid sequences encoding afunctional NAGLU protein described herein are assembled and placed intoany suitable genetic element, e.g., naked DNA, phage, transposon,cosmid, episome, etc., which transfers the NAGLU sequences carriedthereon to a host cell, e.g., for generating non-viral delivery systems(e.g., RNA-based systems, naked DNA, or the like), or for generatingviral vectors in a packaging host cell, and/or for delivery to a hostcells in a subject. In one embodiment, the genetic element is a vector.In one embodiment, the genetic element is a plasmid. The methods used tomake such engineered constructs are known to those with skill in nucleicacid manipulation and include genetic engineering, recombinantengineering, and synthetic techniques. See, e.g., Green and Sambrook,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, NY (2012).

The term “percent (%) identity”, “sequence identity”, “percent sequenceidentity”, or “percent identical” in the context of nucleic acidsequences refers to the residues in the two sequences which are the samewhen aligned for correspondence. The length of sequence identitycomparison may be over the full-length of the genome, the full-length ofa gene coding sequence, or a fragment of at least about 500 to 5000nucleotides, is desired. However, identity among smaller fragments, e.g.of at least about nine nucleotides, usually at least about 20 to 24nucleotides, at least about 28 to 32 nucleotides, at least about 36 ormore nucleotides, may also be desired.

Multiple sequence alignment programs are also available for nucleic acidsequences. Examples of such programs include, “Clustal Omega”, “ClustalW”, “CAP Sequence Assembly”, “BLAST”, “MAP”, and “MEME”, which areaccessible through Web Servers on the internet. Other sources for suchprograms are known to those of skill in the art. Alternatively, VectorNTI utilities are also used. There are also a number of algorithms knownin the art that can be used to measure nucleotide sequence identity,including those contained in the programs described above. As anotherexample, polynucleotide sequences can be compared using Fasta™, aprogram in GCG Version 6.1. Fasta™ provides alignments and percentsequence identity of the regions of the best overlap between the queryand search sequences. For instance, percent sequence identity betweennucleic acid sequences can be determined using Fasta™ with its defaultparameters (a word size of 6 and the NOPAM factor for the scoringmatrix) as provided in GCG Version 6.1, herein incorporated byreference.

Percent identity may be readily determined for amino acid sequences overthe full-length of a protein, polypeptide, about 32 amino acids, about330 amino acids, or a peptide fragment thereof or the correspondingnucleic acid sequence coding sequences. A suitable amino acid fragmentmay be at least about 8 amino acids in length, and may be up to about700 amino acids. Generally, when referring to “identity”, “homology”, or“similarity” between two different sequences, “identity”, “homology” or“similarity” is determined in reference to “aligned” sequences.“Aligned” sequences or “alignments” refer to multiple nucleic acidsequences or protein (amino acids) sequences, often containingcorrections for missing or additional bases or amino acids as comparedto a reference sequence.

Identity may be determined by preparing an alignment of the sequencesand through the use of a variety of algorithms and/or computer programsknown in the art or commercially available (e.g., BLAST, ExPASy; ClustalOmega; FASTA; using, e.g., Needleman-Wunsch algorithm, Smith-Watermanalgorithm). Alignments are performed using any of a variety of publiclyor commercially available Multiple Sequence Alignment Programs. Sequencealignment programs are available for amino acid sequences, e.g., the“Clustal Omega”, “Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”,“MEME”, and “Match-Box” programs. Generally, any of these programs areused at default settings, although one of skill in the art can alterthese settings as needed. Alternatively, one of skill in the art canutilize another algorithm or computer program which provides at leastthe level of identity or alignment as that provided by the referencedalgorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids.Res., “A comprehensive comparison of multiple sequence alignments”,27(13):2682-2690 (1999).

As used herein, “a desired function” refers to an NAGLU enzyme activityat least 5%, about 10%, about 15%, about 20%, about 25%, about 30%,about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,about 100%, or greater than 100% of a healthy control.

As used herein, the phrases “ameliorate a symptom”, “improve a symptom”or any grammatical variants thereof, refer to reversal of an MPSIIIB-related symptoms, showdown or prevention of progression of an MPSIIIB-related symptoms. In one embodiment, the amelioration orimprovement refers to the total number of symptoms in a patient afteradministration of the described composition(s) or use of the describedmethod, which is reduced by about 5%, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95% compared to that before the administration or use. In anotherembodiment, the amelioration or improvement refers to the severity orprogression of a symptom after administration of the describedcomposition(s) or use of the described method, which is reduced by about5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, about 95% compared to that before theadministration or use.

It should be understood that the compositions in the functional NAGLUprotein and NAGLU coding sequence described herein are intended to beapplied to other compositions, regiments, aspects, embodiments andmethods described across the Specification.

2. Expression Cassette

In one aspect, provided is an expression cassette comprising anengineered nucleic acid sequence encoding a functional hNAGLU, and aregulatory sequence which direct expression thereof. In one embodiment,an expression cassette comprising an engineered nucleic acid sequence asdescribed herein which encodes a functional hNAGLU, and a regulatorysequence which direct expression thereof. In one embodiment, the hNAGLUcoding sequence is at least 95% identical to SEQ ID NO: 1. In a furtherembodiment, the hNAGLU coding sequence is SEQ ID NO: 1. In oneembodiment, the regulatory sequence comprises a promoter. In a furtherembodiment, the regulatory sequence comprises a CB7 promoter. In oneembodiment, the regulatory sequence further comprises a chickenbeta-actin intron. In one embodiment, the regulatory sequence furthercomprises a rabbit globin poly A.

As used herein, the term “expression” or “gene expression” refers to theprocess by which information from a gene is used in the synthesis of afunctional gene product. The gene product may be a protein, a peptide,or a nucleic acid polymer (such as a RNA, a DNA or a PNA).

As used herein, an “expression cassette” refers to a nucleic acidpolymer which comprises the coding sequences for a functional hNAGLU,promoter, and may include other regulatory sequences therefor, whichcassette may be packaged into a vector.

As used herein, the term “regulatory sequence”, or “expression controlsequence” refers to nucleic acid sequences, such as initiator sequences,enhancer sequences, and promoter sequences, which induce, repress, orotherwise control the transcription of protein encoding nucleic acidsequences to which they are operably linked.

As used herein, the term “operably linked” refers to both expressioncontrol sequences that are contiguous with the nucleic acid sequenceencoding the functional hNAGLU and/or expression control sequences thatact in trans or at a distance to control the transcription andexpression thereof.

The term “exogenous” as used to describe a nucleic acid sequence orprotein means that the nucleic acid or protein does not naturally occurin the position in which it exists in a chromosome, or host cell. Anexogenous nucleic acid sequence also refers to a sequence derived fromand inserted into the same host cell or subject, but which is present ina non-natural state, e.g. a different copy number, or under the controlof different regulatory elements.

The term “heterologous” as used to describe a nucleic acid sequence orprotein means that the nucleic acid or protein was derived from adifferent organism or a different species of the same organism than thehost cell or subject in which it is expressed. The term “heterologous”when used with reference to a protein or a nucleic acid in a plasmid,expression cassette, or vector, indicates that the protein or thenucleic acid is present with another sequence or subsequence which withwhich the protein or nucleic acid in question is not found in the samerelationship to each other in nature.

In one embodiment, the regulatory sequence comprises a promoter. In oneembodiment, the promoter is a chicken β-actin promoter. In a furtherembodiment, the promoter is a hybrid of a cytomegalovirusimmediate-early enhancer and the chicken β-actin promoter (a CB7promoter). In another embodiment, a suitable promoter may includewithout limitation, an elongation factor 1 alpha (EF1 alpha) promoter(see, e.g., Kim D W et al, Use of the human elongation factor 1 alphapromoter as a versatile and efficient expression system. Gene. 1990 Jul.16; 91(2):217-23), a Synapsin 1 promoter (see, e.g., Kügler S et al,Human synapsin 1 gene promoter confers highly neuron-specific long-termtransgene expression from an adenoviral vector in the adult rat braindepending on the transduced area. Gene Ther. 2003 February;10(4):337-47), a neuron-specific enolase (NSE) promoter (see, e.g., KimJ et al, Involvement of cholesterol-rich lipid rafts ininterleukin-6-induced neuroendocrine differentiation of LNCaP prostatecancer cells. Endocrinology. 2004 February; 145(2):613-9. Epub 2003 Oct.16), or a CB6 promoter (see, e.g., Large-Scale Production ofAdeno-Associated Viral Vector Serotype-9 Carrying the Human SurvivalMotor Neuron Gene, Mol Biotechnol. 2016 January; 58(1):30-6. doi:10.1007/s12033-015-9899-5).

In one embodiment, the expression cassette is designed for expressionand secretion in a human subject. In one embodiment, the expressioncassette is designed for expression in the central nervous system (CNS),including the cerebral spinal fluid and brain. In a further embodiment,the expression cassette is useful for expression in both the CNS and inthe liver. Suitable promoters may be selected, including but not limitedto a constitutive promoter, a tissue-specific promoter or aninducible/regulatory promoter. Example of a constitutive promoter ischicken beta-actin promoter. A variety of chicken beta-actin promotershave been described alone, or in combination with various enhancerelements (e.g., CB7 is a chicken beta-actin promoter withcytomegalovirus enhancer elements; a CAG promoter, which includes thepromoter, the first exon and first intron of chicken beta actin, and thesplice acceptor of the rabbit beta-globin gene; a CBh promoter, S J Grayet al, Hu Gene Ther, 2011 September; 22(9): 1143-1153). Examples ofpromoters that are tissue-specific are well known for liver (albumin,Miyatake et al., (1997) J. Virol., 71:5124-32; hepatitis B virus corepromoter, Sandig et al., (1996) Gene Ther., 3:1002-9; alpha-fetoprotein(AFP), Arbuthnot et al., (1996) Hum. Gene Ther., 7:1503-14), neuron(such as neuron-specific enolase (NSE) promoter, Andersen et al., (1993)Cell. Mol. Neurobiol., 13:503-15; neurofilament light-chain gene,Piccioli et al., (1991) Proc. Natl. Acad. Sci. USA, 88:5611-5; and theneuron-specific vgf gene, Piccioli et al., (1995) Neuron, 15:373-84),and other tissues. Alternatively, a regulatable promoter may beselected. See, e.g., WO 2011/126808B2, incorporated by reference herein.

In one embodiment, the regulatory sequence further comprises anenhancer. In one embodiment, the regulatory sequence comprises oneenhancer. In another embodiment, the regulatory sequence contains two ormore expression enhancers. These enhancers may be the same or may bedifferent. For example, an enhancer may include an Alpha mic/bikenhancer or a CMV enhancer. This enhancer may be present in two copieswhich are located adjacent to one another. Alternatively, the dualcopies of the enhancer may be separated by one or more sequences.

In one embodiment, the regulatory sequence further comprises an intron.In a further embodiment, the intron is a chicken beta-actin intron.Other suitable introns include those known in the art may by a humanβ-globulin intron, and/or a commercially available Promega® intron, andthose described in WO 2011/126808.

In one embodiment, the regulatory sequence further comprises aPolyadenylation signal (polyA). In a further embodiment, the polyA is arabbit globin poly A. See, e.g., WO 2014/151341. Alternatively, anotherpolyA, e.g., a human growth hormone (hGH) polyadenylation sequence, anSV40 polyA, or a synthetic polyA may be included in an expressioncassette.

It should be understood that the compositions in the expression cassettedescribed herein are intended to be applied to other compositions,regiments, aspects, embodiments and methods described across theSpecification.

3. Vector

In one aspect, provided herein is a vector comprising an engineerednucleic acid sequence encoding a functional human NAGLU and a regulatorysequence which direct expression thereof in a target cell. In oneembodiment, the hNAGLU coding sequence is at least 95% identical to SEQID NO: 1. In a further embodiment, the hNAGLU coding sequence is SEQ IDNO: 1.

A “vector” as used herein is a biological or chemical moiety comprisinga nucleic acid sequence which can be introduced into an appropriatetarget cell for replication or expression of said nucleic acid sequence.Examples of a vector includes but not limited to a recombinant virus, aplasmid, Lipoplexes, a Polymersome, Polyplexes, a dendrimer, a cellpenetrating peptide (CPP) conjugate, a magnetic particle, or ananoparticle. In one embodiment, a vector is a nucleic acid moleculeinto which an exogenous or heterologous or engineered nucleic acidencoding a functional hNAGLU may be inserted, which can then beintroduced into an appropriate target cell. Such vectors preferably haveone or more origin of replication, and one or more site into which therecombinant DNA can be inserted. Vectors often have means by which cellswith vectors can be selected from those without, e.g., they encode drugresistance genes. Common vectors include plasmids, viral genomes, and“artificial chromosomes”. Conventional methods of generation,production, characterization or quantification of the vectors areavailable to one of skill in the art.

In one embodiment, the vector is a non-viral plasmid that comprises anexpression cassette described thereof, e.g., “naked DNA”, “naked plasmidDNA”, RNA, and mRNA; coupled with various compositions and nanoparticles, including, e.g., micelles, liposomes, cationic lipid-nucleicacid compositions, poly-glycan compositions and other polymers, lipidand/or cholesterol-based-nucleic acid conjugates, and other constructssuch as are described herein. See, e.g., X. Su et al, Mol.Pharmaceutics, 2011, 8 (3), pp 774-787; web publication: Mar. 21, 2011;WO2013/182683, WO 2010/053572 and WO 2012/170930, all of which areincorporated herein by reference.

In certain embodiments, the vector described herein is a“replication-defective virus” or a “viral vector” which refers to asynthetic or artificial viral particle in which an expression cassettecontaining a nucleic acid sequence encoding a functional hNAGLU ispackaged in a viral capsid or envelope, where any viral genomicsequences also packaged within the viral capsid or envelope arereplication-deficient; i.e., they cannot generate progeny virions butretain the ability to infect target cells. In one embodiment, the genomeof the viral vector does not include genes encoding the enzymes requiredto replicate (the genome can be engineered to be “gutless”—containingonly the nucleic acid sequence encoding NAGLU flanked by the signalsrequired for amplification and packaging of the artificial genome), butthese genes may be supplied during production. Therefore, it is deemedsafe for use in gene therapy since replication and infection by progenyvirions cannot occur except in the presence of the viral enzyme requiredfor replication.

As used herein, a recombinant virus vector is an adeno-associated virus(AAV), an adenovirus, a bocavirus, a hybrid AAV/bocavirus, a herpessimplex virus or a lentivirus.

As used herein, the term “host cell” may refer to the packaging cellline in which a vector (e.g., a recombinant AAV) is produced. A hostcell may be a prokaryotic or eukaryotic cell (e.g., human, insect, oryeast) that contains exogenous or heterologous DNA that has beenintroduced into the cell by any means, e.g., electroporation, calciumphosphate precipitation, microinjection, transformation, viralinfection, transfection, liposome delivery, membrane fusion techniques,high velocity DNA-coated pellets, viral infection and protoplast fusion.Examples of host cells may include, but are not limited to an isolatedcell, a cell culture, an Escherichia coli cell, a yeast cell, a humancell, a non-human cell, a mammalian cell, a non-mammalian cell, aninsect cell, an HEK-293 cell, a liver cell, a kidney cell, a cell of thecentral nervous system, a neuron, a glial cell, or a stem cell.

As used herein, the term “target cell” refers to any target cell inwhich expression of the functional NAGLU is desired. In certainembodiments, the term “target cell” is intended to reference the cellsof the subject being treated for MPS IIIB. Examples of target cells mayinclude, but are not limited to, a liver cell, a kidney cell, a cell ofthe central nervous system, a neuron, a glial cell, and a stem cell. Incertain embodiments, the vector is delivered to a target cell ex vivo.In certain embodiments, the vector is delivered to the target cell invivo.

It should be understood that the compositions in the vector describedherein are intended to be applied to other compositions, regiments,aspects, embodiments and methods described across the Specification.

4. Adeno-associated Virus (AAV)

In one aspect, provided herein is a recombinant AAV (rAAV) comprising anAAV capsid and a vector genome packaged therein. The rAAV is for use inthe treatment of Mucopolysaccharidosis III B (MPS IIIB). The vectorgenome comprises an AAV 5′ inverted terminal repeat (ITR), an engineerednucleic acid sequence encoding a functional hNAGLU as described herein,a regulatory sequence which direct expression of hNAGLU in a targetcell, and an AAV 3′ ITR. In one embodiment, the hNAGLU coding sequenceis at least 95% identical to SEQ ID NO: 1. In a further embodiment, thehNAGLU coding sequence is SEQ ID NO: 1. In one embodiment, theregulatory sequence comprises a promoter. In a further embodiment, theregulatory sequence further comprises an enhancer. In one embodiment,the regulatory sequence further comprises an intron. In one embodiment,the regulatory sequence further comprises a poly A. In certainembodiments, the AAV vector genome comprises the sequence of SEQ ID NO:4 (AAV.CB7.CI.hNAGLUco.RBG), which encodes the hNAGLU protein of SEQ IDNO: 5. In one embodiment, the AAV capsid is an AAV9 capsid. In oneembodiment, the rAAV described herein is for use in the treatment ofMucopolysaccharidosis III B (MPS IIIB).

In one embodiment, the regulatory sequence is as described above. In oneembodiment, the vector genome comprises an AAV 5′ inverted terminalrepeat (ITR), an expression cassette as described herein, and an AAV 3′ITR.

In one embodiment, provided is a rAAV comprising an AAV serotype 9(AAV9) capsid and a vector genome comprising a CB7 promoter expressingan engineered version of hNAGLU with a rabbit beta-globin (rBG) polyAsequence. In a further embodiment, the rAAV vector genome comprises thesequence of SEQ ID NO: 4 (AAV.CB7.CI.hSNAGLUco.rBG). In one embodiment,the rAAV comprises an AAV9 capsid and a vector genome comprising thesequence of SEQ ID NO: 4, wherein the rAAV is represented asAAV9.CB7.CI.hSNAGLUco.rBG.

As used herein, a “vector genome” refers to the nucleic acid sequencepackaged inside a vector. In one embodiment, the vector genome refers tothe nucleic acid sequence packaged inside a rAAV capsid forming an rAAVvector. Such a nucleic acid sequence contains AAV inverted terminalrepeat sequences (ITRs). In one example, a vector genome contains, at aminimum, from 5′ to 3′, an AAV2 5′ ITR, a nucleic acid sequence encodinga functional NAGLU, and an AAV2 3′ ITR. However, ITRs from a differentsource AAV other than AAV2 may be selected. Further, other ITRs may beused. Further, the vector genome contains regulatory sequences whichdirect expression of the functional NAGLU.

The ITRs are the genetic elements responsible for the replication andpackaging of the genome during vector production and are the only viralcis elements required to generate rAAV. In one embodiment, the ITRs arefrom an AAV different than that supplying a capsid. In a preferredembodiment, the ITR sequences from AAV2, or the deleted version thereof(ΔITR), which may be used for convenience and to accelerate regulatoryapproval. However, ITRs from other AAV sources may be selected. Wherethe source of the ITRs is from AAV2 and the AAV capsid is from anotherAAV source, the resulting vector may be termed pseudotyped. Typically,AAV vector genome comprises an AAV 5′ ITR, the NAGLU coding sequencesand any regulatory sequences, and an AAV 3′ ITR. However, otherconfigurations of these elements may be suitable. A shortened version ofthe 5′ ITR, termed ΔITR, has been described in which the D-sequence andterminal resolution site (trs) are deleted. In other embodiments, thefull-length AAV 5′ and 3′ ITRs are used.

The term “AAV” as used herein refers to naturally occurringadeno-associated viruses, adeno-associated viruses available to one ofskill in the art and/or in light of the composition(s) and method(s)described herein, as well as artificial AAVs. An adeno-associated virus(AAV) viral vector is an AAV DNase-resistant particle having an AAVprotein capsid into which is packaged expression cassette flanked by AAVinverted terminal repeat sequences (ITRs) for delivery to target cells.An AAV capsid is composed of 60 capsid (cap) protein subunits, VP1, VP2,and VP3, that are arranged in an icosahedral symmetry in a ratio ofapproximately 1:1:10 to 1:1:20, depending upon the selected AAV. VariousAAVs may be selected as sources for capsids of AAV viral vectors asidentified above. See, e.g., US Published Patent Application No.2007-0036760-A1; US Published Patent Application No. 2009-0197338-A1; EP1310571. See also, WO 2003/042397 (AAV7 and other simian AAV), U.S. Pat.Nos. 7,790,449 and 7,282,199 (AAV8), WO 2005/033321 and U.S. Pat. No.7,906,111 (AAV9), and WO 2006/110689, and WO 2003/042397 (rh.10). Thesedocuments also describe other AAV which may be selected for generatingAAV and are incorporated by reference. Among the AAVs isolated orengineered from human or non-human primates (NHP) and wellcharacterized, human AAV2 is the first AAV that was developed as a genetransfer vector; it has been widely used for efficient gene transferexperiments in different target tissues and animal models. Unlessotherwise specified, the AAV capsid, ITRs, and other selected AAVcomponents described herein, may be readily selected from among any AAV,including, without limitation, the AAVs commonly identified as AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8 bp, AAV7M8 andAAVAnc80, variants of any of the known or mentioned AAVs or AAVs yet tobe discovered or variants or mixtures thereof. See, e.g., WO2005/033321, which is incorporated herein by reference. In oneembodiment, the AAV capsid is an AAV9 capsid or variant thereof. Incertain embodiments, the capsid protein is designated by a number or acombination of numbers and letters following the term “AAV” in the nameof the rAAV vector. In one embodiment, provided is a rAAV(AAV9.CB7.CI.hSNAGLUco.rBG) comprising an AAV serotype 9 (AAV9) capsidand a vector genome comprising the sequence of SEQ ID NO: 4(AAV.CB7.CI.hSNAGLUco.rBG).

As used herein, relating to AAV, the term “variant” means any AAVsequence which is derived from a known AAV sequence, including thosewith a conservative amino acid replacement, and those sharing at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 97%, at least 99% or greater sequence identity over theamino acid or nucleic acid sequence. In another embodiment, the AAVcapsid includes variants which may include up to about 10% variationfrom any described or known AAV capsid sequence. That is, the AAV capsidshares about 90% identity to about 99.9% identity, about 95% to about99% identity or about 97% to about 98% identity to an AAV capsidprovided herein and/or known in the art. In one embodiment, the AAVcapsid shares at least 95% identity with an AAV capsid. When determiningthe percent identity of an AAV capsid, the comparison may be made overany of the variable proteins (e.g., vp1, vp2, or vp3).

The ITRs or other AAV components may be readily isolated or engineeredusing techniques available to those of skill in the art from an AAV.Such AAV may be isolated, engineered, or obtained from academic,commercial, or public sources (e.g., the American Type CultureCollection, Manassas, VA). Alternatively, the AAV sequences may beengineered through synthetic or other suitable means by reference topublished sequences such as are available in the literature or indatabases such as, e.g., GenBank, PubMed, or the like. AAV viruses maybe engineered by conventional molecular biology techniques, making itpossible to optimize these particles for cell specific delivery ofnucleic acid sequences, for minimizing immunogenicity, for tuningstability and particle lifetime, for efficient degradation, for accuratedelivery to the nucleus, etc.

As used herein, the terms “rAAV” and “artificial AAV” usedinterchangeably, mean, without limitation, a AAV comprising a capsidprotein and a vector genome packaged therein, wherein the vector genomecomprising a nucleic acid heterologous to the AAV. In one embodiment,the capsid protein is a non-naturally occurring capsid. Such anartificial capsid may be generated by any suitable technique, using aselected AAV sequence (e.g., a fragment of a vp1 capsid protein) incombination with heterologous sequences which may be obtained from adifferent selected AAV, non-contiguous portions of the same AAV, from anon-AAV viral source, or from a non-viral source. An artificial AAV maybe, without limitation, a pseudotyped AAV, a chimeric AAV capsid, arecombinant AAV capsid, or a “humanized” AAV capsid. Pseudotypedvectors, wherein the capsid of one AAV is replaced with a heterologouscapsid protein, are useful in the invention. In one embodiment, AAV2/5and AAV2/8 are exemplary pseudotyped vectors. The selected geneticelement may be delivered by any suitable method, including transfection,electroporation, liposome delivery, membrane fusion techniques, highvelocity DNA-coated pellets, viral infection and protoplast fusion. Themethods used to make such constructs are known to those with skill innucleic acid manipulation and include genetic engineering, recombinantengineering, and synthetic techniques. See, e.g., Green and Sambrook,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, NY (2012).

As used herein, “AAV9 capsid” refers to the AAV9 having the amino acidsequence of (a) GenBank accession: AAS99264, is incorporated byreference herein and the AAV vp1 capsid protein is reproduced in SEQ IDNO: 6, and/or (b) the amino acid sequence encoded by the nucleotidesequence of GenBank Accession: AY530579.1: (nt 1 . . . 2211) (reproducedin SEQ ID NO: 7). Some variation from this encoded sequence isencompassed by the present invention, which may include sequences havingabout 99% identity to the referenced amino acid sequence in GenBankaccession: AAS99264 and U.S. Pat. No. 7,906,111 (also WO 2005/033321)(i.e., less than about 1% variation from the referenced sequence). SuchAAV may include, e.g., natural isolates (e.g., hu68 (described inco-pending U.S. Patent Applications No. 62/464,748, filed Feb. 28, 2017and U.S. Patent Application No. 62/591,002, filed Nov. 27, 2019, bothentitled “Novel Adeno-associated virus (AAV) Clade F Vector and UsesTherefor” and WO 2018/160582), hu31 or hu32), or variants of AAV9 havingamino acid substitutions, deletions or additions, e.g., including butnot limited to amino acid substitutions selected from alternate residues“recruited” from the corresponding position in any other AAV capsidaligned with the AAV9 capsid; e.g., such as described in U.S. Pat. Nos.9,102,949, 8,927,514, US2015/349911; WO 2016/049230A11; U.S. Pat. Nos.9,623,120; 9,585,971. However, in other embodiments, other variants ofAAV9, or AAV9 capsids having at least about 95% identity to theabove-referenced sequences may be selected. See, e.g., US PublishedPatent Application No. 2015/0079038. Methods of generating the capsid,coding sequences therefore, and methods for production of rAAV viralvectors have been described. See, e.g., Gao, et al, Proc. Natl. Acad.Sci. U.S.A. 100 (10), 6081-6086 (2003) and US 2013/0045186A1.

In one embodiment, the rAAV as described herein is a self-complementaryAAV. “Self-complementary AAV” refers a construct in which a codingregion carried by a recombinant AAV nucleic acid sequence has beendesigned to form an intra-molecular double-stranded DNA template. Uponinfection, rather than waiting for cell mediated synthesis of the secondstrand, the two complementary halves of scAAV will associate to form onedouble stranded DNA (dsDNA) unit that is ready for immediate replicationand transcription. See, e.g., D M McCarty et al, “Self-complementaryrecombinant adeno-associated virus (scAAV) vectors promote efficienttransduction independently of DNA synthesis”, Gene Therapy, (August2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs aredescribed in, e.g., U.S. Pat. Nos. 6,596,535; 7,125,717; and 7,456,683,each of which is incorporated herein by reference in its entirety.

In certain embodiments, the rAAV described herein is nuclease-resistant.Such nuclease may be a single nuclease, or mixtures of nucleases, andmay be endonucleases or exonucleases. A nuclease-resistant rAAVindicates that the AAV capsid has fully assembled and protects thesepackaged genomic sequences from degradation (digestion) during nucleaseincubation steps designed to remove contaminating nucleic acids whichmay be present from the production process. In many instances, the rAAVdescribed herein is DNase resistant.

The recombinant adeno-associated virus (AAV) described herein may begenerated using techniques which are known. See, e.g., WO 2003/042397;WO 2005/033321, WO 2006/110689; U.S. Pat. No. 7,588,772 B2. Such amethod involves culturing a host cell which contains a nucleic acidsequence encoding an AAV capsid; a functional rep gene; an expressioncassette as described herein flanked by AAV inverted terminal repeats(ITRs); and sufficient helper functions to permit packaging of theexpression cassette into the AAV capsid protein. Also provided herein isthe host cell which contains a nucleic acid sequence encoding an AAVcapsid; a functional rep gene; a vector genome as described; andsufficient helper functions to permit packaging of the vector genomeinto the AAV capsid protein. In one embodiment, the host cell is a HEK293 cell. These methods are described in more detail in WO2017160360 A2,which is incorporated by reference herein.

Other methods of producing rAAV available to one of skill in the art maybe utilized. Suitable methods may include without limitation,baculovirus expression system or production via yeast. See, e.g., RobertM. Kotin, Large-scale recombinant adeno-associated virus production. HumMol Genet. 2011 Apr. 15; 20(R1): R2-R6. Published online 2011 Apr. 29.doi: 10.1093/hmg/ddr141; Aucoin M G et al., Production ofadeno-associated viral vectors in insect cells using triple infection:optimization of baculovirus concentration ratios. Biotechnol Bioeng.2006 Dec. 20; 95(6):1081-92; SAMI S. THAKUR, Production of RecombinantAdeno-associated viral vectors in yeast. Thesis presented to theGraduate School of the University of Florida, 2012; Kondratov O et al.Direct Head-to-Head Evaluation of Recombinant Adeno-associated ViralVectors Manufactured in Human versus Insect Cells, Mol Ther. 2017 Aug.10. pii: 51525-0016(17)30362-3. doi: 10.1016/j.ymthe.2017.08.003. [Epubahead of print]; Mietzsch M et al, OneBac 2.0: Sf9 Cell Lines forProduction of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation ofForeign DNA. Hum Gene Ther Methods. 2017 February; 28(1):15-22. doi:10.1089/hgtb.2016.164.; Li L et al. Production and characterization ofnovel recombinant adeno-associated virus replicative-form genomes: aeukaryotic source of DNA for gene transfer. PLoS One. 2013 Aug. 1;8(8):e69879. doi: Print 2013; Galibert L et al, Latest developments inthe large-scale production of adeno-associated virus vectors in insectcells toward the treatment of neuromuscular diseases. J InvertebrPathol. 2011 July; 107 Suppl:S80-93. doi: and Kotin R M, Large-scalerecombinant adeno-associated virus production. Hum Mol Genet. 2011 Apr.15; 20(R1):R2-6. doi: 10.1093/hmg/ddr141. Epub 2011 Apr. 29.

A two-step affinity chromatography purification at high saltconcentration followed by anion exchange resin chromatography are usedto purify the vector drug product and to remove empty capsids. Thesemethods are described in more detail in WO 2017/160360 entitled“Scalable Purification Method for AAV9”, which is incorporated byreference herein. In brief, the method for separating rAAV9 particleshaving packaged genomic sequences from genome-deficient AAV9intermediates involves subjecting a suspension comprising recombinantAAV9 viral particles and AAV 9 capsid intermediates to fast performanceliquid chromatography, wherein the AAV9 viral particles and AAV9intermediates are bound to a strong anion exchange resin equilibrated ata pH of 10.2, and subjected to a salt gradient while monitoring eluatefor ultraviolet absorbance at about 260 and about 280. Although lessoptimal for rAAV9, the pH may be in the range of about 10.0 to 10.4. Inthis method, the AAV9 full capsids are collected from a fraction whichis eluted when the ratio of A260/A280 reaches an inflection point. Inone example, for the Affinity Chromatography step, the diafilteredproduct may be applied to a Capture Select™ Poros-AAV2/9 affinity resin(Life Technologies) that efficiently captures the AAV2/9 serotype. Underthese ionic conditions, a significant percentage of residual cellularDNA and proteins flow through the column, while AAV particles areefficiently captured.

Conventional methods for characterization or quantification of rAAV areavailable to one of skill in the art. To calculate empty and fullparticle content, VP3 band volumes for a selected sample (e.g., inexamples herein an iodixanol gradient-purified preparation where # ofGC=# of particles) are plotted against GC particles loaded. Theresulting linear equation (y=mx+c) is used to calculate the number ofparticles in the band volumes of the test article peaks. The number ofparticles (pt) per 20 μL loaded is then multiplied by 50 to giveparticles (pt)/mL. Pt/mL divided by GC/mL gives the ratio of particlesto genome copies (pt/GC). Pt/mL-GC/mL gives empty pt/mL. Empty pt/mLdivided by pt/mL and ×100 gives the percentage of empty particles.Generally, methods for assaying for empty capsids and AAV vectorparticles with packaged genomes have been known in the art. See, e.g.,Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec.Ther. (2003) 7:122-128. To test for denatured capsid, the methodsinclude subjecting the treated AAV stock to SDS-polyacrylamide gelelectrophoresis, consisting of any gel capable of separating the threecapsid proteins, for example, a gradient gel containing 3-8%Tris-acetate in the buffer, then running the gel until sample materialis separated, and blotting the gel onto nylon or nitrocellulosemembranes, preferably nylon. Anti-AAV capsid antibodies are then used asthe primary antibodies that bind to denatured capsid proteins,preferably an anti-AAV capsid monoclonal antibody, most preferably theB1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Viral. (2000)74:9281-9293). A secondary antibody is then used, one that binds to theprimary antibody and contains a means for detecting binding with theprimary antibody, more preferably an anti-IgG antibody containing adetection molecule covalently bound to it, most preferably a sheepanti-mouse IgG antibody covalently linked to horseradish peroxidase. Amethod for detecting binding is used to semi-quantitatively determinebinding between the primary and secondary antibodies, preferably adetection method capable of detecting radioactive isotope emissions,electromagnetic radiation, or colorimetric changes, most preferably achemiluminescence detection kit. For example, for SDS-PAGE, samples fromcolumn fractions can be taken and heated in SDS-PAGE loading buffercontaining reducing agent (e.g., DTT), and capsid proteins were resolvedon pre-cast gradient polyacrylamide gels (e.g., Novex). Silver stainingmay be performed using SilverXpress (Invitrogen, CA) according to themanufacturer's instructions or other suitable staining method, i.e.SYPRO ruby or coomassie stains. In one embodiment, the concentration ofAAV vector genomes (vg) in column fractions can be measured byquantitative real time PCR (Q-PCR). Samples are diluted and digestedwith DNase I (or another suitable nuclease) to remove exogenous DNA.After inactivation of the nuclease, the samples are further diluted andamplified using primers and a TaqMan™ fluorogenic probe specific for theDNA sequence between the primers. The number of cycles required to reacha defined level of fluorescence (threshold cycle, Ct) is measured foreach sample on an Applied Biosystems Prism 7700 Sequence DetectionSystem. Plasmid DNA containing identical sequences to that contained inthe AAV vector is employed to generate a standard curve in the Q-PCRreaction. The cycle threshold (Ct) values obtained from the samples areused to determine vector genome titer by normalizing it to the Ct valueof the plasmid standard curve. End-point assays based on the digital PCRcan also be used.

In one aspect, an optimized q-PCR method is used which utilizes a broadspectrum serine protease, e.g., proteinase K (such as is commerciallyavailable from Qiagen). More particularly, the optimized qPCR genometiter assay is similar to a standard assay, except that after the DNaseI digestion, samples are diluted with proteinase K buffer and treatedwith proteinase K followed by heat inactivation. Suitably samples arediluted with proteinase K buffer in an amount equal to the sample size.The proteinase K buffer may be concentrated to 2 fold or higher.Typically, proteinase K treatment is about 0.2 mg/mL, but may be variedfrom 0.1 mg/mL to about 1 mg/mL. The treatment step is generallyconducted at about 55° C. for about 15 minutes, but may be performed ata lower temperature (e.g., about 37° C. to about 50° C.) over a longertime period (e.g., about 20 minutes to about 30 minutes), or a highertemperature (e.g., up to about 60° C.) for a shorter time period (e.g.,about 5 to 10 minutes). Similarly, heat inactivation is generally atabout 95° C. for about 15 minutes, but the temperature may be lowered(e.g., about 70 to about 90° C.) and the time extended (e.g., about 20minutes to about 30 minutes). Samples are then diluted (e.g., 1000-fold)and subjected to TaqMan analysis as described in the standard assay.

Additionally, or alternatively, droplet digital PCR (ddPCR) may be used.For example, methods for determining single-stranded andself-complementary AAV vector genome titers by ddPCR have beendescribed. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum GeneTher Methods. 2014 April; 25(2):115-25. doi: 10.1089/hgtb.2013.131. Epub2014 Feb. 14.

Methods for determining the ratio among vp1, vp2 and vp3 of capsidprotein are also available. See, e.g., Vamseedhar Rayaprolu et al,Comparative Analysis of Adeno-Associated Virus Capsid Stability andDynamics, J Virol. 2013 December; 87(24): 13150-13160; Buller R M, RoseJ A. 1978. Characterization of adenovirus-associated virus-inducedpolypeptides in KB cells. J. Virol. 25:331-338; and Rose J A, Maizel JV, Inman J K, Shatkin A J. 1971. Structural proteins ofadenovirus-associated viruses. J. Virol. 8:766-770.

As used herein, the term “treatment” or “treating” refers tocomposition(s) and/or method(s) for the purposes of amelioration of oneor more symptoms of MPS IIIB, restore of a desired function of NAGLU, orimprovement of biomarker of disease. In some embodiments, the term“treatment” or “treating” is defined encompassing administering to asubject one or more compositions described herein for the purposesindicated herein. “Treatment” can thus include one or more of reducingonset or progression of MPS IIIB, preventing disease, reducing theseverity of the disease symptoms, retarding their progression, removingthe disease symptoms, delaying progression of disease, or increasingefficacy of therapy in a given subject.

It should be understood that the compositions in the rAAV describedherein are intended to be applied to other compositions, regiments,aspects, embodiments and methods described across the Specification.

5. Pharmaceutical Composition

In one aspect, provided herein is a pharmaceutical compositioncomprising a vector as described herein in a formulation buffer. In oneembodiment, the pharmaceutical composition is suitable forco-administering with a functional hNAGLU protein or a proteincomprising a functional hNAGLU. In one embodiment, provided is apharmaceutical composition comprising a rAAV as described herein in aformulation buffer. In one embodiment, the rAAV is formulated at about1×10⁹ genome copies (GC)/mL to about 1×10¹⁴ GC/mL. In a furtherembodiment, the rAAV is formulated at about 3×10⁹ GC/mL to about 3×10¹³GC/mL. In yet a further embodiment, the rAAV is formulated at about1×10⁹ GC/mL to about 1×10¹³ GC/mL. In one embodiment, the rAAV isformulated at least about 1×10¹¹ GC/mL.

In one embodiment, the formulation further comprises a surfactant,preservative, excipients, and/or buffer dissolved in the aqueoussuspending liquid. In one embodiment, the buffer is PBS. In anotherembodiment, the buffer is an artificial cerebrospinal fluid (aCSF),e.g., Eliott's formulation buffer; or Harvard apparatus perfusion fluid(an artificial CSF with final Ion Concentrations (in mM): Na 150; K 3.0;Ca 1.4; Mg 0.8; P 1.0; Cl 155). Various suitable solutions are knownincluding those which include one or more of: buffering saline, asurfactant, and a physiologically compatible salt or mixture of saltsadjusted to an ionic strength equivalent to about 100 mM sodium chloride(NaCl) to about 250 mM sodium chloride, or a physiologically compatiblesalt adjusted to an equivalent ionic concentration.

Suitably, the formulation is adjusted to a physiologically acceptablepH, e.g., in the range of pH 6 to 8, or pH 6.5 to 7.5, pH 7.0 to 7.7, orpH 7.2 to 7.8. As the pH of the cerebrospinal fluid is about 7.28 toabout 7.32, for intrathecal delivery, a pH within this range may bedesired; whereas for intravenous delivery, a pH of 6.8 to about 7.2 maybe desired. However, other pHs within the broadest ranges and thesesubranges may be selected for other route of delivery.

A suitable surfactant, or combination of surfactants, may be selectedfrom among non-ionic surfactants that are nontoxic. In one embodiment, adifunctional block copolymer surfactant terminating in primary hydroxylgroups is selected, e.g., such as Pluronic® F68 [BASF], also known asPoloxamer 188, which has a neutral pH, has an average molecular weightof 8400. Other surfactants and other Poloxamers may be selected, i.e.,nonionic triblock copolymers composed of a central hydrophobic chain ofpolyoxypropylene (poly (propylene oxide)) flanked by two hydrophilicchains of polyoxyethylene (poly (ethylene oxide)), SOLUTOL HS 15(Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride),polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acidesters), ethanol and polyethylene glycol. In one embodiment, theformulation contains a poloxamer. These copolymers are commonly namedwith the letter “P” (for poloxamer) followed by three digits: the firsttwo digits×100 give the approximate molecular mass of thepolyoxypropylene core, and the last digit×10 gives the percentagepolyoxyethylene content. In one embodiment Poloxamer 188 is selected.The surfactant may be present in an amount up to about 0.0005% to about0.001% of the suspension.

In one example, the formulation may contain, e.g., buffered salinesolution comprising one or more of sodium chloride, sodium bicarbonate,dextrose, magnesium sulfate (e.g., magnesium sulfate·7H2O), potassiumchloride, calcium chloride (e.g., calcium chloride·2H2O), dibasic sodiumphosphate, and mixtures thereof, in water. Suitably, for intrathecaldelivery, the osmolarity is within a range compatible with cerebrospinalfluid (e.g., about 275 to about 290); see, e.g.,emedicine.medscape.com/article/2093316-overview. Optionally, forintrathecal delivery, a commercially available diluent may be used as asuspending agent, or in combination with another suspending agent andother optional excipients. See, e.g., Elliotts B® solution [LukareMedical].

In other embodiments, the formulation may contain one or more permeationenhancers. Examples of suitable permeation enhancers may include, e.g.,mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate,sodium salicylate, sodium caprylate, sodium caprate, sodium laurylsulfate, polyoxyethylene-9-laurel ether, or EDTA.

Additionally provided is a pharmaceutical composition comprising apharmaceutically acceptable carrier and a vector comprising a nucleicacid sequence encoding a functional NAGLU as described herein. As usedherein, “carrier” includes any and all solvents, dispersion media,vehicles, coatings, diluents, antibacterial and antifungal agents,isotonic and absorption delaying agents, buffers, carrier solutions,suspensions, colloids, and the like. The use of such media and agentsfor pharmaceutical active substances is well known in the art.Supplementary active ingredients can also be incorporated into thecompositions. Delivery vehicles such as liposomes, nanocapsules,microparticles, microspheres, lipid particles, vesicles, and the like,may be used for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the rAAV vector maybe formulated for delivery either encapsulated in a lipid particle, aliposome, a vesicle, a nanosphere, or a nanoparticle or the like. In oneembodiment, a therapeutically effective amount of said vector isincluded in the pharmaceutical composition. The selection of the carrieris not a limitation of the present invention. Other conventionalpharmaceutically acceptable carrier, such as preservatives, or chemicalstabilizers. Suitable exemplary preservatives include chlorobutanol,potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, theparabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.Suitable chemical stabilizers include gelatin and albumin.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a host.

As used herein, the term “dosage” or “amount” can refer to the totaldosage or amount delivered to the subject in the course of treatment, orthe dosage or amount delivered in a single unit (or multiple unit orsplit dosage) administration.

Also, the replication-defective virus compositions can be formulated indosage units to contain an amount of replication-defective virus that isin the range of about 1.0×10⁹ GC to about 1.0×10¹⁶ GC (to treat anaverage subject of 70 kg in body weight) including all integers orfractional amounts within the range, and preferably 1.0×10¹² GC to1.0×10¹⁴ GC for a human patient. In one embodiment, the compositions areformulated to contain at least 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹,7×10⁹, 8×10⁹, or 9×10⁹ GC per dose including all integers or fractionalamounts within the range. In another embodiment, the compositions areformulated to contain at least 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, or 9×10¹⁰ GC per dose including all integers orfractional amounts within the range. In another embodiment, thecompositions are formulated to contain at least 1×10¹¹, 2×10¹¹, 3×10¹¹,4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, or 9×10¹¹ GC per dose includingall integers or fractional amounts within the range. In anotherembodiment, the compositions are formulated to contain at least 1×10¹²,2×10¹², 3×10¹², 4×10¹², 5×10¹², 6×10¹², 7×10¹², 8×10¹², or 9×10¹² GC perdose including all integers or fractional amounts within the range. Inanother embodiment, the compositions are formulated to contain at least1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³, 6×10¹³, 7×10¹³, 8×10¹³, or9×10¹³ GC per dose including all integers or fractional amounts withinthe range. In another embodiment, the compositions are formulated tocontain at least 1×10¹⁴, 2×10¹⁴, 3×10¹⁴, 4×10¹⁴, 5×10¹⁴, 6×10¹⁴, 7×10¹⁴,8×10¹⁴, or 9×10¹⁴ GC per dose including all integers or fractionalamounts within the range. In another embodiment, the compositions areformulated to contain at least 1×10¹⁵, 2×10¹⁵, 3×10¹⁵, 4×10¹⁵, 5×10¹⁵,6×10¹⁵, 7×10¹⁵, 8×10¹⁵, or 9×10¹⁵ GC per dose including all integers orfractional amounts within the range. In one embodiment, for humanapplication the dose can range from 1×10¹⁰ to about 1×10¹² GC per doseincluding all integers or fractional amounts within the range.

In one embodiment, the pharmaceutical composition comprising a rAAV asdescribed herein is administrable at a dose of about 1×10⁹ GC per gramof brain mass to about 1×10¹⁴ GC per gram of brain mass.

The aqueous suspension or pharmaceutical compositions described hereinare designed for delivery to subjects in need thereof by any suitableroute or a combination of different routes. In one embodiment, thepharmaceutical composition is formulated for delivery viaintracerebroventricular (ICV), intrathecal (IT), or intracisternalinjection. In one embodiment, the compositions described herein aredesigned for delivery to subjects in need thereof by intravenousinjection. Alternatively, other routes of administration may be selected(e.g., oral, inhalation, intranasal, intratracheal, intraarterial,intraocular, intramuscular, and other parenteral routes).

As used herein, the terms “intrathecal delivery” or “intrathecaladministration” refer to a route of administration for drugs via aninjection into the spinal canal, more specifically into the subarachnoidspace so that it reaches the cerebrospinal fluid (CSF). Intrathecaldelivery may include lumbar puncture, intraventricular,suboccipital/intracisternal, and/or C1-2 puncture. For example, materialmay be introduced for diffusion throughout the subarachnoid space bymeans of lumbar puncture. In another example, injection may be into thecisterna magna. Intracisternal delivery may increase vector diffusionand/or reduce toxicity and inflammation caused by the administration.See, e.g., Christian Hinderer et al, Widespread gene transfer in thecentral nervous system of cynomolgus macaques following delivery of AAV9into the cisterna magna, Mol Ther Methods Clin Dev. 2014; 1: 14051.Published online 2014 Dec. 10. doi: 10.1038/mtm.2014.51.

As used herein, the terms “intracisternal delivery” or “intracisternaladministration” refer to a route of administration for drugs directlyinto the cerebrospinal fluid of the brain ventricles or within thecisterna magna cerebellomedularis, more specifically via a suboccipitalpuncture or by direct injection into the cisterna magna or viapermanently positioned tube. FIG. 6 provides an illustration as to howan intracisternal injection would be made.

It should be understood that the compositions in the pharmaceuticalcomposition described herein are intended to be applied to othercompositions, regiments, aspects, embodiments and methods describedacross the Specification.

6. Method of Treatment

In one aspect, provided herein is a method of treating a human subjectdiagnosed with MPS IIIB. Currently, when there is a clinical suspicionof MPS III, the first step is the request of a quantitative test todetect the presence of GAGs in urine through spectrophotometric methodsusing dimethylmethylene blue (DMB). The DMB test is based on the unionof GAGs to the dimethylmethylene blue and the quantification of theGAG-DMB complex with a spectrophotometer. The sensitivity of this testis 100%, with a specificity of 75-100%. A negative result when detectingGAGs in urine does not rule out the existence of MPS III due to the factthat in some patients with attenuated forms of the disease, the levelsof GAGs excretion with healthy controls can overlap and the increasedexcretion of heparan sulfate in the MPS III can be ignored. The currentgold standard technique for diagnosis is the determination of enzymeactivity in cultured skin fibroblasts, leukocytes, plasma or serum. Thespecific diagnosis of MPS IIIB is confirmed by showing a decrease orabsence of one of the NAGLU enzymatic activities involved in thedegradation of heparan sulfate in the patient's leukocytes orfibroblasts; the reduction should be less than 10% when compared to theactivity in healthy individuals, with normalcy in other sulfatases.Because the disease due to deficiency in multiple sulfatases also showsa reduction in the activity of the heparan N-sulfatase,N-acetylglucosamine 6-sulfatase and other sulfatases, biochemicalanalysis of at least other sulfatase is required to confirm thediagnosis of MPS III and thus rule out multiple sulfatases deficiency.However, the method of diagnosis is not a limitation of the presentinvention and other suitable methods may be selected.

The method comprises administering to a subject a suspension of a vectoras described herein. In one embodiment, the method comprisesadministering to a subject a suspension of a rAAV as described herein ina formulation buffer at a dose of about 1×10⁹ GC per gram of brain massto about 1×10¹⁴ GC per gram of brain mass.

The composition(s) and method(s) provided achieve efficacy in treating asubject in need with MPS IIIB. Efficacy of the method in a subject canbe shown by assessing (a) an increase in NAGLU enzymatic activity; (b)amelioration of a MPS IIIB symptom; (c) improvement of MPS IIIB-relatedbiomarkers, e.g., GAG levels polyamine (e.g., spermine) levels in thecerebrospinal fluid (CSF), serum, urine and/or other biological samples;or (e) facilitation of any treatment(s) for MPS IIIB. In certainembodiments, efficacy may be determined by monitoring cognitiveimprovement and/or anxiety correction, gait and/or mobility improvement,reduction in tremor frequency and/or severity, reduction inclasping/spasms, improvements in posture, improvements in cornealopacity. Examples of suitable scoring, which is hereby incorporated inthis section. Additionally or alternatively, efficacy of the method maybe predicted based on an animal model. One example of a suitable murinemodel is described in Example 1. In another embodiment, a multiparametergrading scale (see, FIG. 5 incorporated herein by reference) wasdeveloped to evaluate disease correction and response to the MPSIIIAvector therapy described herein in an animal model. Animals are assigneda score based on an assessment of a combination of tremor, posture, furquality, clasping, corneal clouding, and gait/mobility. In certainembodiments, any combination of one or more of these factors may be usedto demonstrate efficacy, alone, or in combination with other factors.See, Burkholder et al. Curr Protoc Mouse Biol. June 2012, 2:145-65;Tumpey et al. J Virol. May 1998, 3705-10; and Guyenet et al. J Vis Exp,May 2010, 39; 1787). Cognitive improvement and anxiety correction oftreated animals is evaluated by assessing movement in an open field(i.e. beam break measurement as described, e.g., in Tatem et al. J VisExp, 2014, (91):51785) and the elevated plus maze assay (as described,e.g., in Walf and Frye, Nat Protoc, 2007, 2(2): 322-328).

As used herein, “facilitation of any treatment(s) for MPS IIIB” or anygrammatical variant thereof, refers to a decreased dosage or a lowerfrequency of a treatment of MPS IIIB in a subject other than thecomposition(s) or method(s) which is/are firstly disclosed in theinvention, compared to that of a standard treatment withoutadministration of the described composition(s) and use of the describedmethod(s).

Examples of suitable treatment facilitated by the composition(s) ormethod(s) described herein might include, but not limited to,

-   -   (a) medications used to relieve symptoms (such as seizures and        sleep disturbances) and improve quality of life;    -   (b) hematopoietic stem cell transplantation, such as bone marrow        transplantation or umbilical cord blood transplantation (see,        e.g., Vellodi A, Young E, New M, Pot-Mees C, Hugh-Jones K. Bone        marrow transplantation for Sanfilippo disease type B. J Inherit        Metab Dis. 1992; 15: 911-8; Garbuzova-Davis, S, Willing, AE,        Desjarlais, T, et al. Transplantation of human umbilical cord        blood cells benefits an animal model of Sanfilippo syndrome        type B. Stem Cells Dev. 2005; 14:384-394; and Garbuzova-Davis,        S, Klasko, S K, and Sanberg, PR. Intravenous administration of        human umbilical cord blood cells in an animal model of MPS        III B. J Comp Neurol. 2009; 515:93-101.);    -   (c) enzyme replacement therapies (ERT) (e.g., via intravenous        administration or intracerebroventricular infusion, see, e.g.,        Aoyagi-Scharber M et al, Clearance of Heparan Sulfate and        Attenuation of CNS Pathology by Intracerebroventricular BMN 250        (NAGLU-IGF2) in Sanfilippo Type B Mice, Mol Ther Methods Clin        Dev. 2017 Jun. 6; 6:43-53. doi:        10.1016/j.omtm.2017.05.009.eCollection 2017 Sep. 15; and Alexion        Pharmaceuticals. Safety, Pharmacokinetics, and        Pharmacodynamics/Efficacy of SBC-103 in MPS IIIB. In:        ClinicalTrialsgov [Internet]. Bethesda: National Library of        Medicine (US). 2000, Available from:        clinicaltrials.gov/show/NCT02324049. NLM identifier:        NCT02324049.);    -   (d) substrate reduction therapy (e.g., treatment with genistein.        Delgadillo V et al. Genistein supplementation in patients        affected by Sanfilippo disease. J Inherit Metab Dis. 2011        October; 34(5):1039-44. doi: 10.1007/s10545-011-9342-4. Epub        2011 May 10; Piotrowska E et al, Two-year follow-up of        Sanfilippo Disease patients treated with a genistein-rich        isoflavone extract: assessment of effects on cognitive functions        and general status of patients. Med Sci Monit. 2011 April;        17(4):CR196-202; and Piotrowska, E et al, Genistin-rich soy        isoflavone extract in substrate reduction therapy for sanfilippo        syndrome: an openlabel, pilot study in 10 pediatric patients.        Curr. Ther. Res. Clin. Exp. 2008; 69: 166-179);    -   (e) chaperone therapy (see, IGF2 in Kan S H, Troitskaya L A,        Sinow C S, Haitz K, Todd A K, Di Stefano A, et al. Insulin-like        growth factor II peptide fusion enables uptake and lysosomal        delivery of alpha-N-acetylglucosaminidase to        mucopolysaccharidosis type IIIB fibroblasts. Biochem J. 2014;        458:281-9; HIRMAb in Boado R J, Lu J Z, Hui E K, Lin H,        Pardridge W M. Insulin Receptor Antibody        alpha-N-Acetylglucosaminidase Fusion Protein Penetrates the        Primate Blood-Brain Barrier and Reduces Glycosoaminoglycans in        Sanfilippo Type B Fibroblasts. Mol Pharm. 2016; 13:1385-92;        CpGH89 inhibitor in Ficko-Blean, E, Stubbs, K A, Nemirovsky, O,        et al. Structural and mechanistic insight into the basis of        mucopolysaccharidosis IIIB. Proc Natl Acad Sci USA. 2008;        105:6560-6565; and Zhao, K W and Neufeld, EF. Purification and        characterization of recombinant human        alpha-N-acetylglucosaminidase secreted by Chinese hamster ovary        cells. Protein Expr Purif. 2000; 19:202-211); and    -   (f) any combination thereof.

In one embodiment, the described method results in the subjectdemonstrating an improvement of biomarkers related to MPS IIIB.

An “increase in NAGLU enzymatic activity” is used interchangeably withthe term “increase in desired NAGLU function”, and refers to a NAGLUactivity at least about 5%, 10%, 15%, 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, orabout 100% of the NAGLU enzyme range for a healthy patient. The NAGLUenzymatic activity might be measured by an assay as described herein. Inone embodiment, the NAGLU enzymatic activity might be measured in theserum, plasma, blood, urine, CSF, or another biological sample. In oneembodiment, administration of the composition as described herein, oruse of the method as described herein, result in an increase in NAGLUenzymatic activity in serum, plasma, saliva, urine or other biologicalsamples. Alternatively, CSF GAG levels and other CSF biomarkers such asspermine levels may be measured to determine therapeutic effect. See.e.g., WO 2017/136533.

Neurocognition can be determined by conventional methods, See. e.g., WO2017/136500 A1. Prevention of neurocognitive decline refers to aslowdown of a neurocognitive decline of the subject administered withthe composition described herein or received the method described hereinby at least about 5%, at least about 20%, at least about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 100% compared to that of a MPS IIIB patient.

As used herein, the terms “biomarker” or “MPS IIIB-related biomarker”refer to presence, concentration, expression level or activity of abiological or chemical molecular in a biological sample of a subjectwhich correlates to progression or development of MPS IIIB in a positiveor negative matter. In one embodiment, the biomarker is GAG levels inthe cerebrospinal fluid (CSF), serum, urine, skin fibroblasts,leukocytes, plasma, or any other biological samples. In anotherembodiment, the biomarker is assessed using clinical chemistry. In yetanother embodiment, the biomarker is liver or spleen volumes. In oneembodiment, the biomarker is the activity of the heparan N-sulfatase,N-acetylglucosamine 6-sulfatase and other sulfatases. In anotherembodiment, the biomarker is spermine level in CSF, serum, or anotherbiological sample. In yet another embodiment, the biomarker is lysosomalenzyme activity in serum, CSF, or another biological sample. In oneembodiment, the biomarker is assessed via magnetic resonance imaging(MRI) of brain. In another embodiment, the biomarker is a neurocognitivescore measured by a neurocognitive developmental test. The phrase“improvement of biomarker” as used herein means a reduction in abiomarker positively correlating to the progression of the disease, oran increase in a biomarker negatively correlating to the progression ofthe disease, wherein the reduction or increase is at least about 5%, atleast about 20%, at least about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about100% compared to that before administration of the composition asdescribed herein or use of the method as described herein.

In one embodiment, the method further comprises detecting or monitoringbiomarkers related to MPS IIIB in the subject prior to initiation oftherapy with therapy provided herein. In one aspect, the methodcomprises detection of a biomarker which is a polyamine (such asspermine) in a sample from a subject (see WO/2017/136533, which isincorporated herein by reference). Thus, in one embodiment, the methodcomprises detecting spermine in a patient sample for purposes ofdiagnosing a patient with MPSIIIB. In another embodiment, spermineconcentration levels in a patient sample are detected to monitor theeffectiveness of a treatment for MPSIIIB using the vector as describedherein. Currently, patients with MPSIIIB are not considered candidatesfor bone marrow transplantation (BMT), Substrate Reduction Therapy (SRT)or enzyme replacement therapy (ERT). However, in certain embodiments, agene therapy patient treated with a vector expressing the NAGLUdescribed herein has, at a minimum, sufficient enzyme expression levelsthat any sub-normal range enzyme levels can be treated with ERT or SRT.Such ERT may be a co-therapy in which the dose of the ERT is monitoredand modulated for months or years post-vector dosing. Additionally oralternatively, a SRT may be a co-therapy in which the dose of the SRT ismonitored and modulated for months or years post-vector dosing.Additionally or alternatively, a chaperone therapy may be a co-therapyin which the dose of the chaperone therapy is monitored and modulatedfor months or years post-vector dosing.

Thus, in one embodiment, the suspension is suitable for co-administeringwith a functional hNAGLU protein or a recombinant protein comprising afunctional NAGLU. In one embodiment, the recombinant protein is a NAGLUfused with insulin-like growth factor 2 (IGF2).

In one embodiment, the suspension is delivered into the subject in needintracerebroventricularly, intrathecally, intracisternaly orintravenously.

In one embodiment, the suspension has a pH of about 7.28 to about 7.32.

As used herein, an enzyme replacement therapy (ERT) is a medicaltreatment that consists in replacing an enzyme in patients where aparticular enzyme is deficient or absent. The enzyme is usually producedas a recombinant protein and administrated to the patient. In oneembodiment, the enzyme is a functional NAGLU. In another embodiment, theenzyme is a recombinant protein comprising a functional NAGLU. In oneembodiment, the enzyme is a recombinant protein comprising a functionalNAGLU and an insulin-like growth factor 2 (IGF2). Aoyagi-Scharber M etal, Clearance of Heparan Sulfate and Attenuation of CNS Pathology byIntracerebroventricular BMN 250 in Sanfilippo Type B Mice, Mol TherMethods Clin Dev. 2017 Jun. 6; 6:43-53. doi: eCollection 2017 Sep. 15;and WO2017132675A1. Systemic, intrathecal, intracerebroventricular orintracisternal delivery can be used for ERT or SRT co-therapy.

As used herein, an Substrate Reduction Therapy (SRT) refers to a therapyusing a small molecule drug to partially inhibit the biosynthesis of thecompounds, which accumulate in the absence of NAGLU. In one embodiment,the SRT is a therapy via genistein. See, e.g., Ritva Tikkanen et al,Less Is More: Substrate Reduction Therapy for Lysosomal StorageDisorders. Int J Mol Sci. 2016 July; 17(7): 1065. Published online 2016Jul. 4. doi: 10.3390/ijms17071065; Delgadillo V et al, Epub 2011 May 10;and de Ruijter J et al, Genistein in Sanfilippo disease: a randomizedcontrolled crossover trial. Ann Neurol. 2012 January; 71(1):110-20. doi:10.1002/ana.22643. NAGLU.

As used herein, a chaperone therapy refers to a therapy using a smallmolecule drug to helps folding and/or secretion of NAGLUE. In oneembodiment, the chaperone therapy is a therapy via IGF2. See, e.g., KanS H, Troitskaya L A, Sinow C S, Haitz K, Todd A K, Di Stefano A, et al.Insulin-like growth factor II peptide fusion enables uptake andlysosomal delivery of alpha-N-acetylglucosaminidase tomucopolysaccharidosis type IIIB fibroblasts. Biochem J. 2014; 458:281-9;and HIRMAb in Boado R J, Lu J Z, Hui E K, Lin H, Pardridge W M. InsulinReceptor Antibodyalpha-N-Acetylglucosaminidase Fusion Protein Penetratesthe Primate Blood-Brain Barrier and Reduces Glycosoaminoglycans inSanfilippo Type B Fibroblasts. Mol Pharm. 2016; 13:1385-92. In anotherembodiment, the chaperone therapy is a therapy via CpGH89 inhibitor.See, e.g., Ficko-Blean, E, Stubbs, KA, Nemirovsky, O, et al. Structuraland mechanistic insight into the basis of mucopolysaccharidosis IIIB.Proc Natl Acad Sci USA. 2008; 105:6560-6565. In yet another embodiment,the chaperone therapy is a therapy disclosed in Zhao, K W and Neufeld,EF. Purification and characterization of recombinant humanalpha-N-acetylglucosaminidase secreted by Chinese hamster ovary cells.Protein Expr Purif. 2000; 19:202-211.

Suitable volumes for delivery of these doses and concentrations may bedetermined by one of skill in the art. For example, volumes of about 1μL to 150 mL may be selected, with the higher volumes being selected foradults. Typically, for newborn infants a suitable volume is about 0.5 mLto about 10 mL, for older infants, about 0.5 mL to about 15 mL may beselected. For toddlers, a volume of about 0.5 mL to about 20 mL may beselected. For children, volumes of up to about 30 mL may be selected.For pre-teens and teens, volumes up to about 50 mL may be selected. Instill other embodiments, a patient may receive an intrathecaladministration in a volume of about 5 mL to about 15 mL are selected, orabout 7.5 mL to about 10 mL. Other suitable volumes and dosages may bedetermined. The dosage will be adjusted to balance the therapeuticbenefit against any side effects and such dosages may vary dependingupon the therapeutic application for which the recombinant vector isemployed.

In one embodiment, the rAAV as described herein is administrable at adose of about 1×10⁹ GC per gram of brain mass to about 1×10¹⁴ GC pergram of brain mass. In certain embodiments, the rAAV is co-administeredsystemically at a dose of about 1×10⁹ GC per kg body weight to about1×10¹³ GC per kg body weight.

In one embodiment, the subject is delivered a therapeutically effectiveamount of the vectors described herein. As used herein, a“therapeutically effective amount” refers to the amount of thecomposition comprising the nucleic acid sequence encoding a functionalNAGLU which delivers and expresses in the target cells an amount ofenzyme sufficient to achieve efficacy. In one embodiment, the dosage ofthe vector is about 1×10⁹ GC per gram of brain mass to about 1×10¹³genome copies (GC) per gram (g) of brain mass, including all integers orfractional amounts within the range and the endpoints. In anotherembodiment, the dosage is 1×10¹⁰ GC per gram of brain mass to about1×10¹³ GC per gram of brain mass. In specific embodiments, the dose ofthe vector administered to a patient is at least about 1.0×10⁹ GC/g,about 1.5×10⁹ GC/g, about 2.0×10⁹ GC/g, about 2.5×10⁹ GC/g, about3.0×10⁹ GC/g, about 3.5×10⁹ GC/g, about 4.0×10⁹ GC/g, about 4.5×10⁹GC/g, about 5.0×10⁹ GC/g, about 5.5×10⁹ GC/g, about 6.0×10⁹ GC/g, about6.5×10⁹ GC/g, about 7.0×10⁹ GC/g, about 7.5×10⁹ GC/g, about 8.0×10⁹GC/g, about 8.5×10⁹ GC/g, about 9.0×10⁹ GC/g, about 9.5×10⁹ GC/g, about1.0×10¹⁰ GC/g, about 1.5×10¹⁰ GC/g, about 2.0×10¹⁰ GC/g, about 2.5×10¹⁰GC/g, about 3.0×10¹⁰ GC/g, about 3.5×10¹⁰ GC/g, about 4.0×10¹⁰ GC/g,about 4.5×10¹⁰ GC/g, about 5.0×10¹⁰ GC/g, about 5.5×10¹⁰ GC/g, about6.0×10¹⁰ GC/g, about 6.5×10¹⁰ GC/g, about 7.0×10¹⁰ GC/g, about 7.5×10¹⁰GC/g, about 8.0×10¹⁰ GC/g, about 8.5×10¹⁰ GC/g, about 9.0×10¹⁰ GC/g,about 9.5×10¹⁰ GC/g, about 1.0=10¹¹ GC/g, about 1.5×10¹¹ GC/g, about2.0×10¹¹ GC/g, about 2.5×10¹¹ GC/g, about 3.0×10¹¹ GC/g, about 3.5×10¹¹GC/g, about 4.0×10¹¹ GC/g, about 4.5×10¹¹ GC/g, about 5.0×10¹¹ GC/g,about 5.5×10¹¹ GC/g, about 6.0×10¹¹ GC/g, about 6.5×10¹¹ GC/g, about7.0×10¹¹ GC/g, about 7.5×10¹¹ GC/g, about 8.0×10¹¹ GC/g, about 8.5×10¹¹GC/g, about 9.0×10¹¹ GC/g, about 9.5×10¹¹ GC/g, about 1.0×10¹² GC/g,about 1.5×10¹² GC/g, about 2.0×10¹² GC/g, about 2.5×10¹² GC/g, about3.0×10¹² GC/g, about 3.5×10¹² GC/g, about 4.0×10¹² GC/g, about 4.5×10¹²GC/g, about 5.0×10¹² GC/g, about 5.5×10¹² GC/g, about 6.0×10¹² GC/g,about 6.5×10¹² GC/g, about 7.0×10¹² GC/g, about 7.5×10¹² GC/g, about8.0×10¹² GC/g, about 8.5×10¹² GC/g, about 9.0×10¹² GC/g, about 9.5×10¹²GC/g, about 1.0×10¹³ GC/g, about 1.5×10¹³ GC/g, about 2.0×10¹³ GC/g,about 2.5×10¹³ GC/g, about 3.0×10¹³ GC/g, about 3.5×10¹³ GC/g, about4.0×10¹³ GC/g, about 4.5×10¹³ GC/g, about 5.0×10¹³ GC/g, about 5.5×10¹³GC/g, about 6.0×10¹³ GC/g, about 6.5×10¹³ GC/g, about 7.0×10¹³ GC/g,about 7.5×10¹³ GC/g, about 8.0×10¹³ GC/g, about 8.5×10¹³ GC/g, about9.0×10¹³ GC/g, about 9.5×10¹³ GC/g, or about 1.0×10¹⁴ GC/g brain mass.

In one embodiment, the method further comprises the subject receives animmunosuppressive co-therapy. Immunosuppressants for such co-therapyinclude, but are not limited to, a glucocorticoid, steroids,antimetabolites, T-cell inhibitors, a macrolide (e.g., a rapamycin orrapalog), and cytostatic agents including an alkylating agent, ananti-metabolite, a cytotoxic antibiotic, an antibody, or an agent activeon immunophilin. The immune suppressant may include a nitrogen mustard,nitrosourea, platinum compound, methotrexate, azathioprine,mercaptopurine, fluorouracil, dactinomycin, an anthracycline, mitomycinC, bleomycin, mithramycin, IL-2 receptor-(CD25-) or CD3-directedantibodies, anti-IL-2 antibodies, ciclosporin, tacrolimus, sirolimus,IFN-β, IFN-γ, an opioid, or TNF-α (tumor necrosis factor-alpha) bindingagent.

In certain embodiments, the immunosuppressive therapy may be started 0,1, 2, 7, or more days prior to the gene therapy administration. Suchtherapy may involve co-administration of two or more drugs, the (e.g.,prednelisone, micophenolate mofetil (MMF) and/or sirolimus (i.e.,rapamycin)) on the same day. One or more of these drugs may be continuedafter gene therapy administration, at the same dose or an adjusted dose.Such therapy may be for about 1 week (7 days), about 60 days, or longer,as needed. In certain embodiments, a tacrolimus-free regimen isselected.

In certain embodiment, the method comprises measurement of serumanti-hNAGLU antibodies. Suitable assays of measuring anti-hNAGLUantibody are available, See, e.g., Example 1.

In one embodiment, the rAAV as described herein is administrated once tothe subject in need. In another embodiment, the rAAV is administratedmore than once to the subject in need.

It should be understood that the compositions in the method describedherein are intended to be applied to other compositions, regiments,aspects, embodiments and methods described across the Specification.

7. Kit

In certain embodiments, a kit is provided which includes a concentratedvector suspended in a formulation (optionally frozen), optional dilutionbuffer, and devices and components required for intrathecal,intracerebroventricular or intracisternal administration. In anotherembodiment, the kit may additional or alternatively include componentsfor intravenous delivery. In one embodiment, the kit provides sufficientbuffer to allow for injection. Such buffer may allow for about a 1:1 toa 1:5 dilution of the concentrated vector, or more. In otherembodiments, higher or lower amounts of buffer or sterile water areincluded to allow for dose titration and other adjustments by thetreating clinician. In still other embodiments, one or more componentsof the device are included in the kit. Suitable dilution buffer isavailable, such as, a saline, a phosphate buffered saline (PBS) or aglycerol/PBS.

It should be understood that the compositions in kit described hereinare intended to be applied to other compositions, regiments, aspects,embodiments and methods described across the Specification.

8. Device

In one aspect, the vectors provided herein may be administeredintrathecally via the method and/or the device described, e.g., in WO2017/136500, which is incorporated herein by reference in its entirety.Alternatively, other devices and methods may be selected. In summary,the method comprises the steps of advancing a spinal needle into thecisterna magna of a patient, connecting a length of flexible tubing to aproximal hub of the spinal needle and an output port of a valve to aproximal end of the flexible tubing, and after said advancing andconnecting steps and after permitting the tubing to be self-primed withthe patient's cerebrospinal fluid, connecting a first vessel containingan amount of isotonic solution to a flush inlet port of the valve andthereafter connecting a second vessel containing an amount of apharmaceutical composition to a vector inlet port of the valve. Afterconnecting the first and second vessels to the valve, a path for fluidflow is opened between the vector inlet port and the outlet port of thevalve and the pharmaceutical composition is injected into the patientthrough the spinal needle, and after injecting the pharmaceuticalcomposition, a path for fluid flow is opened through the flush inletport and the outlet port of the valve and the isotonic solution isinjected into the spinal needle to flush the pharmaceutical compositioninto the patient. This method and this device may each optionally beused for intrathecal delivery of the compositions provided herein.Alternatively, other methods and devices may be used for suchintrathecal delivery.

It should be understood that the compositions in the device describedherein are intended to be applied to other compositions, regiments,aspects, embodiments and methods described across the Specification.

EXAMPLES

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples but rather should be construed to encompass any and allvariations that become evident as a result of the teaching providedherein.

Example 1: Methods

A. Vector-AAV9.CB7.CI. hNAGLUco.rBG

A hNAGLU codon-optimized sequence as shown in SEQ ID NO: 1 was clonedinto an expression construct containing a CB7 promoter (a hybrid of acytomegalovirus immediate-early enhancer and the chicken β-actinpromoter), chicken β-actin intron (CI), and rabbit beta globin (rBG)polyadenylation sequence. The expression construct was flanked by AAV2inverted terminal repeats and an AAV9 trans plasmid was used forencapsidation.

AAV vectors were manufactured by Penn Vector Core with iodixanolgradient method. See, Lock, M., et al., Rapid, Simple, and VersatileManufacturing of Recombinant Adeno-Associated Viral Vectors at Scale.Human Gene Therapy, 2010. 21(10): p. 1259-1271. The purified vectorswere titrated with classic qPCR for MPS IIIB by Penn Vector Core.

Dubelco's phosphate buffer saline (dPBS) without calcium and magnesiumwas used as control article (vehicle control) and diluent for vector.The test article was diluted with sterile phosphate buffered saline(PBS) to the appropriate concentration for each dose group. Dilutedvector was kept on wet ice and injected to the animals within 4 hoursafter dilution.

B. Animal Procedures

All animal protocols were approved by the Institutional Animal Care andUse Committee of the University of Pennsylvania. NAGLU knock-out micewere maintained in the Gene Therapy Program vivarium at the Universityof Pennsylvania. All offspring were genotyped by PCR analysis of tailsnip DNA using an automated system (Transnetyx Inc, 8110 Cordova RoadSuite 119 Cordova, TN 38016). Mice were grouped based on their genotypeafter weaning and were not mixed after that to prevent fighting; allanimals in a given cage received the same treatment.

Animals were housed in standard caging of 1-5 animals/cage under 12-hourlight/dark cycle controlled via automatic timer with a humidity of30-70%. Temperature was kept within the range of 64-79° F. (18-26° C.).Autoclaved rodent chow food was provided ad libitum. Water wasaccessible to all animals ad libitum via individual placed water bottlesin each cage. At a minimum, water bottles were replaced once per weekduring weekly cage changes. The water supply was drawn from the City ofPhiladelphia and purified using a Getinge water purifier. Water qualityis tested by ULAR daily for chlorine levels and quarterly for pH andhardness. Nesting material (Nestlet (D) was provided in each cage aftereach change. Animals were monitored daily by GTP staff and ULARveterinary staff.

C. Vector and Vehicle Administration

MPS IIIB mice vector doses were 3×10 8, 3×10⁹ or 3×10¹⁰ GC per mouse atan average age of 18 weeks. It is noted that ddPCR (Lock, M., et al.,Absolute Determination of Single-Stranded and Self-ComplementaryAdeno-Associated Viral Vector Genome Titers by Droplet Digital PCR.Human Gene Therapy Methods, 2014. 25(2): p. 115-125) gives titers thatare approximately 3 fold higher than the classic qPCR method. Mice wereanesthetized with Isoflurane. Each anesthetized mouse was grasped firmlyby the loose skin behind the head and injected free hand anterior andlateral to the bregma with a Hamilton syringe fitted with a 27-gaugeneedle, which was adjusted to be inserted 3 mm deep.

D. Neurobehavioral Assessment

Rocking rotarod was performed to assess coordination and balance 2months pi (MPS IIIB). Mice were habituated to the rotarod during 2trials at a constant low speed (5 rpm) for 120 seconds. After 2 minutesrest, mice were placed back on the rotarod and submitted to a rockingparadigm were the rod rotates at a constant speed of rpm with reversalof the rotation direction every other rotation. 3 trials were performedwith intertrial rest of 2 minutes. Results were expressed as the averagelatency to fall from the rod; the longer the latency, the better thecoordination.

E. Histology

Mice were euthanized by cardiac puncture exsanguination underketamine/xylazine anesthesia 3 months post injection. Tissues werepromptly collected, half was snap-frozen on dry ice (enzyme activity),and half was immersion-fixed in 10% neutral formalin and embedded inparaffin for histology. Collected tissues were brain, spinal cord,liver, and heart.

Hematoxylin & eosin (H&E) staining was performed according to standardprotocols on paraffin sections. Histopathology was scored in brain andspinal cord by a board-certified veterinary Pathologist blinded to thetreatment. Brain score was the cumulative sum of 4-grade severity scoresof glial cell vacuolation in brain, neuronal vacuolation in braincortex, neuronal vacuolation in brainstem and hindbrain, perivascularmononuclear cell infiltration mononuclear cell infiltration (maximumscore of 20). Cumulative scores were analyzed by one-way Anova KruskallWallis test with post hoc Dunn's multiple comparison test, alpha 0.05.

Lysosomal storage was assessed by LIMP2 immunostaining andquantification. LIMP2 immunostaining was performed on 6 μm sections fromformalin-fixed paraffin-embedded brain tissue. Sections weredeparaffinized through an ethanol and xylene series, boiled in amicrowave for 6 minutes in 10 mmol/L citrate buffer (pH 6.0) for antigenretrieval, and blocked with 1% donkey serum in PBS+0.2% Triton for 15minutes followed by sequential incubation with primary (1 hour) andlabeled secondary (45 minutes) antibodies diluted in blocking buffer.The primary antibody was rabbit anti-LIMP2 (Novus Biologicals,Littleton, CO, 1:200) and the secondary antibody was FITC- orTRITC-labeled donkey anti-rabbit (Jackson Immunoresearch). The number ofcells staining positive for LIMP2 was quantified in 2-4 brain sectionsfrom each animal (Day 90 necropsies) by trained GTP Morphology corepersonnel.

F. Enzyme Activity and Glycosaminoglycan Storage

For enzyme activity assays and GAGs content, proteins were extracted bymechanical homogenization (Qiagen TissueLizer) in an acidic lysissolution (0.2% triton, 0.9% NaCl, adjusted to pH 4). Samples werefreeze-thawed and clarified by centrifugation. Protein was quantified byBCA assay.

NAGLU activity was measured by incubating 10 μL sample with 20 μL of 2mM 4-MU-2-Acetamido-2-deoxy-alpha-D-glucopyranoside (Toronto ResearchChemicals) dissolved in sodium acetate 0.1M pH 3.58; NaCl 150 mM; TritonX100 After incubating for 2 h at 37° C., the mixture was diluted inglycine NaOH buffer, pH 10.6, and released 4-MU was quantified byfluorescence (excitation 365 nm, emission 450 nm) compared with standarddilutions of free 4-MU and normalized by the protein content.

GAGs content in tissue extract is measured using dye-binding method witha commercial kit used per manufacturer recommendations (Blyscan BiocolorGAGs kit).

G. Anti-Transgene Antibodies

Blood for measurement of serum anti-hNAGLU antibodies was collected atseveral in vivo timepoints by submandibular bleeding as well as atterminal necropsy by cardiac puncture. Serum was separated and frozen ondry ice and stored at −80° C. until analyzed. Polystyrene plates werecoated overnight with recombinant human NAGLU (R&D Systems), 5 μg/mL inPBS, titrated to pH 5.8. Plates were washed and blocked 1 hour in 2%bovine serum albumin (BSA) in neutral PBS. Plates were then incubatedwith serum samples diluted 1:1000 in PBS. Bound antibody was detectedwith horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody(Abcam) diluted 1:10,000 in PBS with 2% BSA. The assay was developedusing tetramethylbenzidine substrate and stopped with 2N sulfuric acidbefore measuring absorbance at 450 nm.

Example 2: Determination of Minimum Effective Dose (MED) in a MurineModel of MPSIIIb

Experiments were performed to evaluate the expression, bioactivity, andminimum effective dose (MED) of a single intracerebroventricular (ICV)administration of AAV9.CB7.CI.hNAGLUco.rBG, an AAV9 vector expressinghuman N-acetyl-α-D-glucosaminidase (NAGLU), in a murine model ofMPSIIIb.

AAV9.CB7.CI.hNAGLUco.rBG was administered through the ICV route to MPSIIIb mice, average age of 4 months (n=10 per group) at doses of 3×10⁸ GCor 3×10⁹ GC or 3×10¹⁰ GC (determined by qPCR tittering of the vector) onDay 0 with a 3 month post-injection (pi) observation period. Vehicletreated MPS IIIb and heterozygous littermates served as controls (n=10per group).

Bioactivity was assessed by measuring the NAGLU activity at 3 months piin the brain, spinal cord, liver, serum and heart. Efficacy and MED weredetermined by measuring performance on a rocking rotarod at 2 months pias well as brain and spinal cord lysosomal storage and histopathology at3 month pi.

ICV administration of AAV9.CB7.CI.hNAGLUco.rBG to MPS IIIb mice at up to3×10¹⁰ GC was well tolerated, with no treatment related clinical signsor mortality, and resulted in NAGLU expression in the whole CNS (brainand spinal cord) as well as in peripheral tissues (liver, heart andserum).

There were dose dependent increases in NAGLU activity in the brain,spinal cord, heart and liver at 3 month pi (FIG. 1 ) with enzymaticactivity 50% of the heterozygous level at the mid dose in the brain andsimilar to or above the heterozygous level at high dose in all organsthat were assessed. There was dose dependent normalization of thelysosomal compartment, as shown by reductions in LIMP2 staining in thebrain at the high dose 3 months pi (FIGS. 2A and 2B). In H&E stainedbrain sections, dose dependent reductions in the amount and frequency ofglial and neuronal vacuolation, indicators of lysosomal storage, wereobserved at the mid and high doses (FIGS. 3A and 3B). Neuronal storageand white matter axonopathy were corrected in the spinal cord at thehigh dose only (not shown). Corresponding to the changes in CNSlysosomal content and improvements in disease-related morphology in theH&E stained sections, there were improvement in the balance andcoordination assessed by the rocking rotarod assay at 2 month pi at thehigh dose only, the statistical significance was however not reached dueto interindividual variability (FIG. 4 ).

The test article and injection procedure were well tolerated. Noclinical abnormality was noted in the mice apart from the MPs IIIbphenotype related signs. All mice survived up to the scheduledeuthanasia. There was no evidence of test-article related toxicity inthe brain on histopathology, although changes related to the ICVadministration procedure itself were observed in some mice (focalhemosiderophages and mononuclear cell infiltrates in the periventricularparenchyma and meninges).

In conclusion, AAV9.CB7.CI.hNAGLUco.rBG was well tolerated in MPS IIIbmice at all dose levels and resulted in dose-dependent increases inNAGLU levels (expression and enzymatic activity) that were associatedwith improvements in both CNS and peripheral parameters of MPS IIIb withimprovement of the neurobehavioral phenotype at the high dose. The highdose administered, 3×10¹⁰ GC, was the minimum effective dose (MED) inthis study (for neurobehavioral rescue) and the mid dose, 3×10⁹ GC, wasthe MED if we consider enzymatic and pathology rescue of the brain. Thetreatment was administered relatively late (4 months) when the storageand neuroinflammation are already well developed, probably explainingthe apparent lack of efficacy of the mid dose despite partial enzymaticrescue in the brain. The mid dose is anticipated to provide behavioralrescue if administered earlier.

Example 3: Pharmacology/Toxicology Study in Rhesus Macaque

Experiments are performed to evaluate the safety of intrathecaladministration of three doses of AAV9.CB7.CI.hNAGLUco.rBG.

The control article is administered via suboccipital puncture to 3macaques (both genders) in Group 1. The vector ofAAV9.CB7.CI.hNAGLUco.rBG is administered via suboccipital puncture to 9rhesus macaques randomized to Groups 2-4. Macaques in Group 3 receivetest article high dose (N=3); macaques in Group 3 receive test articlemiddle dose (N=3); macaques in Group 4 receive test article low dose(N=3). Blood and cerebrospinal fluid are collected as part of a generalsafety panel. Serum and peripheral blood mononuclear cells (PBMC) arecollected to investigate humoral and cellular immune response to thecapsid and transgene.

Following completion of the in-life phase of these studies at 90±3 dayspost-vector administration, macaques are necropsied with tissuesharvested for a comprehensive histopathological examination. Lymphocytesare harvested from spleen, and bone marrow to examine the presence ofCTLs in these organs at the time of necropsy.

Example 3: Long Term Effects of AAV.hNAGLU Administration

Experiments are performed to investigate the long-term effects ofAAV.hNAGLU on MPS IIIb mice. Twenty MPS IIIb mice are injected with ahigh dose of AAV9.CB7.CI.hNAGLU.rBG (9×10¹⁰ GC, ICV) at 2 months of age.An additional twenty MPS Ma mice and twenty wild-type mice receive PBScontrol injections. The mice are monitored for 7 months post injection,during which they are assigned clinical scores weekly and undergobehavioral and cognitive testing.

A multiparameter grading scale was developed to evaluate diseasecorrection and response to treatment for the duration of the study. Ascore is assigned to individual mice based on an assessment of acombination of tremor, posture, fur quality, clasping, corneal clouding,and gait/mobility (FIG. 5 ). The clinical scoring system was adaptedbased on previously described methods (see, e.g., Burkholder et al. CurrProtoc Mouse Biol. June 2012, 2:145-65; Tumpey et al. J Virol. May 1998,3705-10; and Guyenet et al. J Vis Exp, May 2010, 39; 1787).

All publications cited in this specification are incorporated herein byreference in their entireties as is U.S. Provisional Patent ApplicationNo. 62/593,090, filed Nov. 30, 2017. Similarly, the SEQ ID NOs which arereferenced herein and which appear in the appended Sequence Listing areincorporated by reference. While the invention has been described withreference to particular embodiments, it will be appreciated thatmodifications can be made without departing from the spirit of theinvention. Such modifications are intended to fall within the scope ofthe appended claims.

1. A pharmaceutical composition comprising a plurality of recombinantAAV (rAAV) comprising an AAV capsid and a vector genome packagedtherein, wherein the vector genome comprises an AAV 5′ inverted terminalrepeat (ITR), an engineered nucleic acid sequence encoding a functionalhuman N-acetyl-alpha-glucosaminidase (hNAGLU), a regulatory sequencewhich directs expression of hNAGLU in a target cell, and an AAV 3′ ITR,wherein the hNAGLU coding sequence is at least 95% identical to SEQ IDNO:
 1. 2. The pharmaceutical composition according to claim 1, whereinthe hNAGLU coding sequence is SEQ ID NO:1.
 3. The pharmaceuticalcomposition according to claim 1, wherein the AAV vector genomecomprises the sequence of SEQ ID NO:
 4. 4. The pharmaceuticalcomposition according to claim 1, wherein the AAV capsid is an AAV9capsid.
 5. The pharmaceutical composition according to claim 1 furthercomprising a formulation buffer.
 6. The pharmaceutical compositionaccording to claim 5, which is formulated for delivery viaintracerebroventricular (ICV), intrathecal (IT), intracisternal orintravenous (IV) injection.
 7. A method of treating a human subjectdiagnosed with MPS IIIB and/or improving gait or mobility, reducingtremors, reducing spasms, improving posture, or reducing the progressionof vision loss in a subject in need thereof, comprising administering tothe subject a composition comprising the rAAV in a dose of 1×10⁹ GC pergram of brain mass to about 1×10¹³ GC per gram of brain mass.
 8. Themethod according to claim 7, wherein said method results in a serumNAGLU activity at least about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about100% of a healthy control.
 9. The method according to claim 7, whereinthe suspension is suitable for co-administering with a functional hNAGLUprotein.
 10. The method according to claim 7, wherein the suspension isdelivered into the subject in need intracerebroventricularly,intrathecally, or intravenously.
 11. A nucleic acid molecule comprisingan engineered nucleic acid sequence encoding a functional hNAGLU and aregulatory sequence which directs expression thereof in a target cell,wherein the hNAGLU coding sequence is SEQ ID NO: 1 or at least 95%identical to SEQ ID NO:
 1. 12. The nucleic acid molecule according toclaim 11, wherein the hNAGLU coding sequence is SEQ ID NO:
 1. 13. Thenucleic acid molecule according to claim 11, which is a plasmid.
 14. Acell comprising the nucleic acid molecule according to claim
 11. 15. Thecell according to claim 14, wherein the cell is a yeast cell, a humancell, a non-human cell, a mammalian cell, a non-mammalian cell, or aninsect cell.
 16. The cell according to claim 14 which is an HEK-293cell.